USE OF EphA4 AND MODULATOR OF EphA4 FOR DIAGNOSIS, TREATMENT AND PREVENTION OF CANCER

ABSTRACT

The present invention relates to methods and compositions designed for the treatment, management, or prevention of cancer, particularly, metastatic cancer. In one embodiment, the methods of the invention comprise the administration of an effective amount of one or more antibodies that bind to EphA4 and agonize EphA4. In another embodiment, the methods of the invention comprise the administration of an effective amount of one or more antibodies that bind to EphA4 and inhibit cancer cell colony formation in soft agar or tubular network formation in three-dimensional basement membrane or extracellular matrix preparation. In another embodiment, the methods of the invention comprise the administration of an effective amount of one or more antibodies that preferentially binds to an EphA4 epitope that is exposed on cancer cells but not non-cancer cells. In another embodiment, the methods of the invention comprise the administration of an effective amount of one or more antibodies that bind to EphA4 with a very low K off  to reduce EphA4 expression and, thereby, inhibit tumor cell growth and/or metastasis. The invention also provides pharmaceutical compositions comprising one or more EphA4 antibodies of the invention either alone or in combination with one or more other agents useful for cancer therapy.

This application claims priority to U.S. Provisional Patent applicationNo. 60/476,909, filed Jun. 6, 2003, and U.S. Provisional Patentapplication No. 60/503,356, filed Sep. 16, 2003, each of which isincorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions designed forthe treatment, management, or prevention of hyperproliferative celldisease, particularly cancer. The methods of the invention comprise theadministration of an effective amount of one or more antibodies specificfor EphA4, preferably monoclonal antibodies, that are EphA4 agonists,inhibit a cancer cell phenotype (such as colony formation in soft agaror tubular network formation in a three dimensional basement membrane orextracellular membrane preparation, such as MATRIGEL™), preferentiallybind epitopes on EphA4 that are selectively exposed or increased oncancer cells but not non-cancer cells and/or bind EphA4 with a K_(off)of less than 3×10⁻³ s⁻¹. The invention also provides pharmaceuticalcompositions comprising one or more monoclonal antibodies of theinvention either alone or in combination with one or more other agentsuseful for cancer therapy. Diagnostic methods and methods for screeningfor therapeutically useful EphA4 specific antibodies are also provided.

2. BACKGROUND OF THE INVENTION Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, Basic Pathology,2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arisein many sites of the body and behave differently depending upon itsorigin. Cancerous cells destroy the part of the body in which theyoriginate and then spread to other part(s) of the body where they startnew growth and cause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and, if current trendscontinue, cancer is expected to be the leading cause of death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime.

A cure for cancer has yet to be found. Current treatment options, suchas surgery, chemotherapy and radiation treatment, are often eitherineffective or present serious side effects.

Metastasis

The most life-threatening forms of cancer often arise when a populationof tumor cells gains the ability to colonize distant and foreign sitesin the body. These metastatic cells survive by overriding restrictionsthat normally constrain cell colonization into dissimilar tissues. Forexample, typical mammary epithelial cells will generally not grow orsurvive if transplanted to the lung, yet lung metastases are a majorcause of breast cancer morbidity and mortality. Recent evidence suggeststhat dissemination of metastatic cells through the body can occur longbefore clinical presentation of the primary tumor. These micrometastaticcells may remain dormant for many months or years following thedetection and removal of the primary tumor. Thus, a better understandingof the mechanisms that allow for the growth and survival of metastaticcells in a foreign microenvironment is critical for the improvement oftherapeutics designed to fight metastatic cancer and diagnostics for theearly detection and localization of metastases.

Cancer Cell Signaling

Cancer is a disease of aberrant signal transduction. Aberrant cellsignaling overrides anchorage-dependent constraints on cell growth andsurvival (Rhim, et al., Critical Reviews in Oncogenesis 8:305, 1997;Patarca, Critical Reviews in Oncogenesis 7:343, 1996; Malik, et al.,Biochimica et Biophysica Acta 1287:73, 1996; Cance, et al., BreastCancer Res Treat 35:105, 1995). Tyrosine kinase activity is induced byECM anchorage and indeed, the expression or function of tyrosine kinasesis usually increased in malignant cells (Rhim, et al., Critical Reviewsin Oncogenesis 8:305,1997; Cance, et al., Breast Cancer Res Treat35:105, 1995; Hunter, Cell 88:333, 1997). Based on evidence thattyrosine kinase activity is necessary for malignant cell growth,tyrosine kinases have been targeted with new therapeutics (Levitzki, etal., Science 267:1782, 1995; Kondapaka, et al., Molecular & CellularEndocrinology 117:53, 1996; Fry, et al., Current Opinion inBioTechnology 6: 662, 1995). Unfortunately, obstacles associated withspecific targeting to tumor cells often limit the application of thesedrugs. In particular, tyrosine kinase activity is often vital for thefunction and survival of benign tissues (Levitzki, et al., Science267:1782, 1995). To minimize collateral toxicity, it is critical toidentify and then target tyrosine kinases that are selectivelyoverexpressed in tumor cells.

EphA4

EphA4 is a receptor tyrosine kinase that is expressed in brain, heart,lung, muscle, kidney, placenta, pancreas (Fox, et al, Oncogene 10:897,1995) and melanocytes (Easty, et al., Int. J. Cancer 71:1061, 1997).EphA4 binds cell membrane-anchored ligands (Ephrins A1, A2, A3, A4, A5,B2, and B3; Pasquale, Curr. Opin. in Cell Biology, 1997, 9:608; alsoligands B61, AL1/RAGS, LERK4, Htk-L, and Elk-L3; Martone, et al., BrainResearch 771:238, 1997), and ligand binding leads to EphA4autophosphorylation on tyrosine residues (Ellis, et al., Oncogene12:1727, 1996). EphA4 tyrosine phosphorylation creates a binding regionfor proteins with Src Homology 2/3 (SH2/SH3) domains, such as thecytoplasmic tyrosine kinase p59fyn (Ellis, et al., supra; Cheng, et al.,Cytokine and Growth Factor Reviews 13:75, 2002). Activation of EphA4 inXenopus embryos leads to loss of cadherin-dependent cell adhesion(Winning, et al., Differentiation 70:46, 2002; Cheng, et al., supra),suggesting a role for EphA4 in tumor angiogenesis; however, the role ofEphA4 in cancer progression is unclear. EphA4 appears to be upregulatedin breast cancer, esophageal cancer, and pancreatic cancer (Kuang, etal., Nucleic Acids Res. 26:1116, 1998; Meric, et al, Clinical CancerRes. 8:361, 2002; Nemoto, et al., Pathobiology 65:195, 1997; Logsdon, etal., Cancer Res. 63:2649, 2003), yet it is downregulated in melanomatissue (Easty, et al., supra).

Cancer Therapy

One barrier to the development of anti-metastasis agents has been theassay systems that are used to design and evaluate these drugs. Mostconventional cancer therapies target rapidly growing cells. However,cancer cells do not necessarily grow more rapidly but instead surviveand grow under conditions that are non-permissive to normal cells(Lawrence and Steeg, 1996, World J. Urol. 14:124-130). These fundamentaldifferences between the behaviors of normal and malignant cells provideopportunities for therapeutic targeting. The paradigm thatmicrometastatic tumors have already disseminated throughout the bodyemphasizes the need to evaluate potential chemotherapeutic drugs in thecontext of a foreign and three-dimensional microenvironment. Manystandard cancer drug assays measure tumor cell growth or survival undertypical cell culture conditions (i.e., monolayer growth). However, cellbehavior in two-dimensional assays often does not reliably predict tumorcell behavior in vivo.

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (see, for example, Stockdale, 1998, “Principles of CancerPatient Management”, in Scientific American: Medicine, vol. 3,Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancertherapy may also involve biological therapy or immunotherapy. All ofthese approaches can pose significant drawbacks for the patient.Surgery, for example, may be contraindicated due to the health of thepatient or may be unacceptable to the patient. Additionally, surgery maynot completely remove the neoplastic tissue. Radiation therapy is onlyeffective when the neoplastic tissue exhibits a higher sensitivity toradiation than normal tissue, and radiation therapy can also oftenelicit serious side effects. Hormonal therapy is rarely given as asingle agent and, although it can be effective, is often used to preventor delay recurrence of cancer after other treatments have removed themajority of the cancer cells. Biological therapies/immunotherapies arelimited in number and each therapy is generally effective for a veryspecific type of cancer.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly, or indirectly by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (see, for example, Gilman et al., Goodmanand Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.(Pergamom Press, New York, 1990)). These agents, which includealkylating agents, such as nitrosourea, anti-metabolites, such asmethotrexate and hydroxyurea, and other agents, such as etoposides,campathecins, bleomycin, doxorubicin, daunorubicin, etc., although notnecessarily cell cycle specific, kill cells during S phase because oftheir effect on DNA replication. Other agents, specifically colchicineand the vinca alkaloids, such as vinblastine and vincristine, interferewith microtubule assembly resulting in mitotic arrest. Chemotherapyprotocols generally involve administration of a combination ofchemotherapeutic agents to increase the efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (see, for example, Stockdale, 1998,“Principles Of Cancer Patient Management” in Scientific AmericanMedicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10).Almost all chemotherapeutic agents are toxic, and chemotherapy causessignificant, and often dangerous, side effects, including severe nausea,bone marrow depression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. Further, it is uncommon for cancerto be treated by only one method. Thus, there is a need for developmentof new therapeutic agents for the treatment of cancer and new, moreeffective, therapy combinations for the treatment of cancer.

3. SUMMARY OF THE INVENTION

EphA4 is over expressed in a number of cancers. The inventors have foundthat EphA4 antibody binding to EphA4 can decrease proliferation and/ormetastatic behavior of a cell, and causes phosphorylation of EphA4 andan EphA4-associated protein of approximately 75 kilodaltons (kDa). Thepresent inventors believe that EphA4 may play a role in tumorigenesisand metastasis similar to that of EphA2. Antibodies that agonize EphA2,i.e., elicit EphA2 signaling, actually decrease EphA2 expression andinhibit tumor cell growth and/or metastasis, as described in co-pendingU.S. patent application Ser. No. 10/436,782, entitled “EphA2 MonoclonalAntibodies and Methods of Use Thereof” filed May 12, 2003 as AttorneyDocket No. 10271-097-999. The inventors have also discovered thatantibody binding of EphA4 is sufficient to decrease cell-ECM attachmentsand induce cell rounding. Cell-ECM attachments are generally understoodto provide physical attachments and intracellular signaling that governmany aspects of cell behavior, including decisions regarding cellgrowth, migration, invasion and differentiation. Thus, EphA4 antibodybinding to EphA4 correlates with increased EphA4 phosphorylation anddecreased cell-ECM attachment. Although not intending to be bound by anymechanism of action, antibodies or other molecules that agonize EphA4may repress hyperproliferation or malignant cell behavior by inducingEphA4 autophosphorylation, thereby causing subsequent EphA4 degradationto down-regulate expression. Thus, in one embodiment, the EphA4antibodies of the invention agonize EphA4 signaling and increasephosphorylation of EphA4 and the EphA4-associated 75 kDa protein (“EphA4agonistic antibodies”).

In addition, cancer cells exhibit phenotypic traits that differ fromthose of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or weblike matrices in a three-dimensional basementmembrane or extracellular matrix preparation, such as MATRIGEL™.Non-cancer cells do not form colonies in soft agar and form distinctsphere-like structures in three-dimensional basement membrane orextracellular matrix preparations. The present inventors have also foundthat anti-EphA4 antibodies can reduce growth of EphA4-expressing cancercells in soft agar. Accordingly, the invention also provides antibodiesthat specifically bind EphA4 and inhibit one or more cancer cellphenotypes, such as colony formation in soft agar or tubular networkformation in three-dimensional basement membrane or extracellular matrixpreparations (“cancer cell phenotype inhibitory EphA4 antibodies”).Exposing cancer cells to such cancer cell phenotype inhibitory EphA4antibodies prevents or decreases the cells' ability to colonize or formtubular networks in these substrates. Furthermore, in certainembodiments, the addition of such cancer cell phenotype inhibitory EphA4antibodies to already established colonies of cancer cells cause areduction or elimination of an existing cancer cell colony, i.e., leadsto killing of hyperproliferative and/or metastatic cells, for examplethrough necrosis or apoptosis.

Differences in the subcellular localization, ligand binding propertiesor protein organization (e.g., structure, orientation in the cellmembrane) may further distinguish the EphA4 that is present on cancercells from EphA4 on non-cancer cells. In non-cancer cells, EphA4 isexpressed at low levels. However, cancer cells generally displaydecreased cell-cell contacts and this may decrease EphA4-ligand binding.Furthermore, the overexpression of EphA4 may cause an excess of EphA4relative to ligand that increases the amount of non-ligand bound EphA4.Consequently, changes in the subcellular distribution or membraneorientation of EphA4 may cause EphA4 to localize to sites in a cancercell where it is inaccessible to ligand. Additionally, EphA4 may havealtered ligand binding properties (e.g., due to an altered conformation)in cancer cells such that it is incapable of stable interactions withits ligand whether or not it is localized to the cell-cell junction. Ineach case, these changes can expose certain epitopes on the EphA4 incancer cells that are not exposed in non-cancer cells. Accordingly, theinvention also provides antibodies that specifically bind EphA4 butpreferably bind an EphA4 epitope exposed on cancer cells but not onnon-cancer cells (“exposed EphA4 epitope antibodies”). Exposing cancercells to such EphA4 antibodies that preferentially bind epitopes onEphA4 that are selectively exposed or increased on cancer cells but notnon-cancer cells targets the therapeutic/prophylactic antibody to cancercells and prevents or decreases the cells' ability to proliferate whilesparing non-cancer cells.

Antibodies that bind EphA4 with a very low K_(off) rate may beparticularly effective to reduce EphA4 expression and/or induce EphA4degradation and, thereby, inhibit tumor cell growth and/or metastasisand/or proliferation of hyperproliferative cells. Accordingly, theinvention further provides antibodies that bind EphA4 with a K_(off) ofless than 3×10⁻³ s⁻¹ and, preferably, are EphA4 agonists.

The present invention provides for the screening and identification ofantibodies that bind to EphA4 and agonize EphA4, inhibit a cancer cellphenotype, preferentially bind epitopes on EphA4 that are selectivelyexposed or increased on cancer cells but not non-cancer cells and/orhave a K_(off) less than 3×10⁻³ s⁻¹, preferably monoclonal antibodies.In particular, the antibodies of the invention bind to the extracellulardomain of EphA4 and, preferably, elicit EphA4 signaling and EphA4autophosphorylation, inhibit a cancer cell phenotype, preferentiallybind an EphA4 epitope exposed on cancer cells but not non-cancer cells,and/or have a K_(off) of less than 3×10⁻³ s⁻¹. The antibodies of theinvention may further be used to modulate EphA4 signaling andphosphorylation of EphA4-associated proteins, such as a 75 kDa proteinthat is phosphorylated on tyrosine residues following crosslinking ofEphA4 with EphA4 antibodies. Antibodies that modulate phosphorylation ofthis 75 kDa EphA4-associated protein can have value as therapeuticagents. In a specific embodiment, the invention provides the EphA4 scFvantibody EphA4-44/EA44 (in the specification herein, EA44 and EphA4-44refer to the same antibody, that is, the EphA4-44 scFv clone which bindsto EphA4 and was deposited with the ATCC on Jun. 10, 2004 as “EA44”). Ina preferred embodiment, the invention provides an EphA4 antibody whereinthe variable heavy chain and/or variable light chain amino acid sequencepossesses at least 90% sequence identity with or is identical to thevariable heavy chain or light chain amino acid sequence of EA44, ascontained SEQ IDs NO: 4 and 8, respectively. In other preferredembodiments, the invention provides an EphA4 antibody wherein at leastthree, at least four, at least five, or all six of its CDRs areidentical to the corresponding CDRs in EA44. In other specificembodiments, the invention provides anti-EphA4 scFv clones 8, 18, 20,36, and 41.

In particular, the invention provides an antibody comprising (oralternatively, consisting of) the EA44 VH CDR1 (SEQ ID NO. 22) and VLCDR1 (SEQ ID NO. 28); the EA44 VH CDR1 (SEQ ID NO. 22) and VL CDR2 (SEQID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22) and VL CDR3 (SEQ ID NO.32); the EA44 VH CDR2 (SEQ ID NO. 24) and VL CDR1 (SEQ ID NO. 28); theEA44 VH CDR2 (SEQ ID NO. 24) and VL CDR2 (SEQ ID NO. 30); the EA44 VHCDR2 (SEQ ID NO. 24) and VH CDR3 (SEQ ID NO. 26); the EA44 VH CDR3 (SEQID NO. 26) and VH CDR1 (SEQ ID NO. 22); the EA44 VH CDR3 (SEQ ID NO. 26)and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR3 (SEQ ID NO. 26) and VLCDR3 (SEQ ID NO. 32); the EA44 VH1 CDR1, VH CDR2 (SEQ ID NO. 24) and VLCDR1 (SEQ ID NO. 28); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ IDNO. 24) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22),VH CDR2 (SEQ ID NO. 24) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR2(SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26) and VL CDR1 (SEQ ID NO. 28),the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26) and VL CDR2(SEQ ID NO. 30); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR2 (SEQ ID NO.24) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1 (SEQ ID NO. 22), VLCDR1 (SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQID NO. 22), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); theEA44 VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQID NO. 30); the EA44 VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28)and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR3 (SEQ ID NO. 26), VL CDR1(SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR3 (SEQ IDNO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO.26) and VL CDR1 (SEQ ID NO. 28); the EA44 VH CDR1 (SEQ ID NO. 22), VHCDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26) and VL CDR2 (SEQ ID NO.30); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3(SEQ ID NO. 26) and VL CDR3 (SEQ ID NO. 32); the EA44VH CDR1 (SEQ ID NO.22), VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24),VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1(SEQ ID NO. 22), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28) and VLCDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR3 (SEQ IDNO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO.28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR2 (SEQ ID NO. 24), VHCDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO.32); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR2(SEQ ID NO. 30) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1 (SEQ IDNO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO.22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ IDNO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1 (SEQ ID NO. 22),VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28), VL CDR2 (SEQ ID NO.30), and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1 (SEQ ID NO. 22), VHCDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28), VL CDR2 (SEQ ID NO. 30),and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3(SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28), VL CDR2 (SEQ ID NO. 30), andVL CDR3 (SEQ ID NO. 32); or any combination thereof of the EA44 VH CDRsand VL CDRs listed in FIG. 7.

In another embodiment, an EphA4 agonistic antibody comprises a VH CDRencoded by a nucleic acid sequence having a nucleotide sequence of SEQID NO. 21, 23, or 25. In another embodiment, an EphA4 agonistic antibodycomprises a VL CDR encoded by a nucleic acid sequence having anucleotide sequence of SEQ ID NO. 27, 29, or 30. In another embodiment,an EphA4 agonistic antibody comprises a VH CDR and a VL CDR encoded by anucleic acid sequence having a nucleotide sequence of SEQ ID NO. 21, 23,or 25, and SEQ ID NO. 27, 29, or 30, respectively.

Accordingly, the present invention relates to pharmaceuticalcompositions and prophylactic and therapeutic regimens designed toprevent, treat, or manage a disease associated with overexpression ofEphA4, particularly cancer, particularly metastatic cancer, in a subjectcomprising administering one or more antibodies that specifically bindto and agonize EphA4, inhibit a cancer cell phenotype (such as colonyformation in soft agar or tubular network formation in a threedimensional basement membrane or extracellular membrane preparation,such as MATRIGEL™), preferentially bind epitopes on EphA4 that areselectively exposed or increased on cancer cells but not non-cancercells and/or have a K_(off) less than 3×10⁻³ s⁻¹. In preferredembodiments, the EphA4 antibody decreases the size of colonies alreadyformed in soft agar and/or reduces the extent of tubular networkformation in a three-dimensional basement membrane or extracellularmatrix preparation. In one embodiment, the cancer is of an epithelialcell origin. In another embodiment, the cancer is a cancer of the skin,lung, colon, prostate, breast, or bladder, or is a renal cell carcinomaor a melanoma. In a preferred embodiment, the cancer is a cancer of thepancreas. In another preferred embodiment, the cancer cells in thecancer to be prevented, treated, or managed overexpress EphA4. In apreferred embodiment, some EphA4 is not bound to ligand, either as aresult of decreased cell-cell contacts, altered subcellularlocalization, or increases in amount of EphA4 relative to ligand. Infurther preferred embodiments, the EphA4 agonist is an EphA4 antibodywherein the variable heavy and/or variable light sequence possesses atleast 90% sequence identity with the variable heavy or light sequence ofEA44, as contained SEQ IDs NO: 22, 24, 26, 28, 30, and 32 respectively.In other preferred embodiments, the invention provides an EphA4 antibodywherein at least three, at least four, at least five, or all six of itsCDRs are identical to the corresponding CDRs in EA44. In other specificembodiments, the invention provides anti-EphA4 scFv clones 8, 18, 20,36, and 41.

In a preferred embodiment, the methods of the invention are used toprevent, treat, or manage metastasis of tumors. The antibodies of theinvention can be administered in combination with one or more othercancer therapies. In particular, the present invention provides methodsof preventing, treating, or managing cancer in a subject comprisingadministering to said subject a therapeutically or prophylacticallyeffective amount of one or more EphA4 antibodies of the invention incombination with the administration of a therapeutically orprophylactically effective amount of one or more chemotherapies,hormonal therapies, biological therapies/immunotherapies and/orradiation therapies other than the administration of an EphA4 antibodyof the invention or in combination with surgery. In a preferredembodiment, one or more EphA4 antibodies are administered in combinationwith one or more EphA2 antibodies.

In other embodiments, the EphA4 antibodies of the invention are used totreat, prevent and/or manage a disease or disorder associated with cellhyperproliferation, such as but not limited to cancer, asthma, chronicobstructive pulmonary disease, inflammatory diseases of the bowel,intestine, stomach, and other vital organs, restenosis (smooth muscleand/or endothelial), Crohn's disease, psoriasis, and othernon-metastatic diseases. In preferred embodiments, thehyperproliferative cells are epithelial. In preferred embodiments, thehyperproliferative cells overexpress EphA4. In a preferred embodiment,some EphA4 is not bound to ligand, either as a result of decreasedcell-cell contacts, altered subcellular localization, or increases inamount of EphA4 relative to EphA4-ligand.

The methods and compositions of the invention are useful not only inuntreated patients but are also useful in the treatment of patientspartially or completely refractory to current standard and experimentalcancer therapies, including but not limited to chemotherapies, hormonaltherapies, biological therapies, radiation therapies, and/or surgery aswell as to improve the efficacy of such treatments. Accordingly, in apreferred embodiment, the invention provides therapeutic andprophylactic agonists for the treatment or prevention of cancer that hasbeen shown to be or may be refractory or non-responsive to therapiesother than those comprising administration of EphA4 antibodies of theinvention. In a specific embodiment, one or more EphA4 antibodies of theinvention are administered to a patient refractory or non-responsive toa non-EphA4-based treatment, particularly tamoxifen treatment or atreatment in which resistance is associated with increased IL-6 levels,to render the patient non-refractory or responsive. The treatment towhich the patient had previously been refractory or non-responsive canthen be administered with therapeutic effect.

In addition, the present invention provides methods of screening forEphA4 antibodies of the invention. In particular, antibodies may bescreened for binding to EphA4, particularly the extracellular domain ofEphA4, using routine immunological techniques. In one embodiment, toidentify agonistic EphA4 antibodies, EphA4 antibodies may be screenedfor the ability to elicit EphA4 signaling, e.g., increase EphA4phosphorylation and/or phosphorylation of a 75 kDa EphA4-associatedprotein and/or to degrade EphA4.

In another embodiment, to identify cancer cell phenotype inhibitingantibodies, anti-EphA4 antibodies may be screened for the ability toprevent or reduce cancer cell colony formation in soft agar or reduce orinhibit tubular network formation in a three-dimensional basementmembrane or extracellular matrix preparation, or any other method thatdetects a decrease in a cancer phenotype, for example, any assay thatdetects an increase in contact inhibition of cell proliferation (e.g.,reduction of colony formation in a monolayer cell culture). In preferredembodiments, the antibodies are screened for ability to decrease thesize of existing colonies in soft agar or reduce the extent or tubularmatrix formation in the three-dimensional basement membrane orextracellular matrix preparation, particular induces cell (particularlycancer cell, more particularly metastatic cancer cell, but alsoincluding other hyperproliferative cell) necrosis or apoptosis.Additionally, antibodies may be screened for their ability to inhibit orreduce colony formation in soft agar and/or tubular network formation inthree-dimensional basement membrane or extracellular matrix preparationsin the presence of other anti-cancer agents, e.g., hormonal,chemotherapeutic, biologic or other anti-cancer agents.

In another embodiment, to identify antibodies that preferentially bindan EphA4 epitope exposed on cancer cells but not non-cancer cells,antibodies may be screened for the ability to preferentially bind EphA4that is not bound to ligand, e.g., Ephrin A4, and that is not localizedto cell-cell contacts. Any method known in the art to determine antibodybinding/localization on a cell can be used to screen candidateantibodies for desirable binding properties. In a specific embodiment,immunofluorescence microscopy or flow cytometry is used to determine thebinding characteristics of an antibody. In this embodiment, antibodiesthat bind poorly to EphA4 when it is bound to ligand and localized tocell-cell contacts but bind well to free EphA4 on a cell are encompassedby the invention. In another specific embodiment, EphA4 antibodies areselected for their ability to compete with ligands (e.g., cell-anchoredor purified ligands) for binding to EphA4 using cell-based or ELISAassays.

In another embodiment, antibodies are screened using antibody bindingkinetic assays well known in the art (e.g. surface plasmon resonancebased assays, such as a BIACORE™ assay) to identify antibodies having aK_(off) rate less than 3×10⁻³ s⁻¹.

In other embodiments, the invention provides methods of treating,preventing, or managing cancer, by administering therapeutic agents,other than EphA4 antibodies of the invention, that reduce EphA4 proteinlevels, for example but not by way of limitation, anti-sense nucleicacids specific for EphA4, double stranded EphA4 RNA that mediates RNAinterference of EphA4 expression, anti-EphA4 ribozymes, etc., as well asother inhibitors of EphA4, for example, small molecule inhibitors ofEphA4.

The invention further provides diagnostic methods using the EphA4antibodies of the invention to evaluate the efficacy of cancertreatment, either EphA4-based or not EphA4-based. In general, increasedEphA4 expression may be associated with increasingly invasive andmetastatic cancers. Accordingly, a reduction in EphA4 expression with aparticular treatment indicates that the treatment is reducing theinvasiveness and/or metastatic potential of cancer. The diagnosticmethods of the invention may also be used to prognose or predict thecourse of cancer or the outcomes of cancer therapy. In particularembodiments, the diagnostic methods of the invention provide methods ofimaging and localizing metastases and methods of diagnosis and prognosisusing tissues and fluids distal to the primary tumor site (as well asmethods using tissues and fluids of the primary tumor), for example,whole blood, sputum, urine, serum, fine needle aspirates (i.e.,biopsies). In other embodiments, the diagnostic methods of the inventionprovide methods of imaging and localizing metastases and methods ofdiagnosis and prognosis in vivo. In such embodiments, primary metastatictumors are detected using an antibody of the invention, preferably anexposed EphA4 epitope antibody. The antibodies of the invention may alsobe used for immunohistochemical analyses of frozen or fixed cells ortissue assays. In addition, the antibodies and diagnostic methods of theinvention may be used to diagnose, prognose or monitor therapy of(whether EphA4 or non-EphA4-based therapy) non-cancer hyperproliferativediseases (particularly associated with EphA4 overexpression), forexample, but not limited to, asthma, psoriasis, inflammatory boweldisease, restenosis, Crohn's disease, prostatic intraepithelialneoplasia (PIN), chronic obstructive pulmonary disease, etc.

In another embodiment, kits comprising the pharmaceutical compositionsor diagnostic reagents of the invention are provided.

3.1 Definitions

As used herein, the term “agonist” refers to any compound including aprotein, polypeptide, peptide, antibody, antibody fragment, largemolecule, or small molecule (less than 10 kD), that increases theactivity, activation or function of another molecule. EphA4 agonistscause increased phosphorylation of EphA4 and/or the 75 kDaEphA4-associated protein, and degradation of EphA4 protein. EphA4antibodies that agonize EphA4 may or may not also inhibit cancer cellphenotype (e.g., colony formation in soft agar or tubular networkformation in a three-dimensional basement membrane or extracellularmatrix preparation) and may or may not preferentially bind an EphA4epitope that is exposed in a cancer cell relative to a non-cancer celland may or may not have a low K_(off) rate.

The term “antibodies or fragments thereof that immunospecifically bindto EphA4” as used herein refers to antibodies or fragments thereof thatspecifically bind to an EphA4 polypeptide or a fragment of an EphA4polypeptide and do not specifically bind to other polypeptides.Preferably, antibodies or fragments that immunospecifically bind to anEphA4 polypeptide or fragment thereof do not non-specificallycross-react with other antigens (e.g., binding cannot be competed awaywith a non-EphA4 protein, e.g., BSA). Antibodies or fragments thatimmunospecifically bind to an EphA4 polypeptide can be identified, forexample, by immunoassays or other techniques known to those of skill inthe art. Antibodies of the invention include, but are not limited to,synthetic antibodies, monoclonal antibodies, recombinantly producedantibodies, intrabodies, multispecific antibodies (including bi-specificantibodies), human antibodies, humanized antibodies, chimericantibodies, synthetic antibodies, single-chain Fvs (scFv), Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (includingbi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. In particular, antibodiesof the present invention include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds to an EphA4 antigen (e.g., one or more complementarity determiningregions (CDRs) of an anti-EphA4 antibody). Preferably agonisticantibodies or fragments that immunospecifically bind to an EphA4polypeptide or fragment thereof preferentially agonize EphA4 and do notsignificantly agonize other activities.

As used herein, the term “cancer” refers to a disease involving cellsthat have the potential to metastasize to distal sites and exhibitphenotypic traits that differ from those of non-cancer cells, forexample, formation of colonies in a three-dimensional substrate such assoft agar or the formation of tubular networks or weblike matrices in athree-dimensional basement membrane or extracellular matrix preparation,such as MATRIGEL™. Non-cancer cells do not form colonies in soft agarand form distinct sphere-like structures in three-dimensional basementmembrane or extracellular matrix preparations. Cancer cells acquire acharacteristic set of functional capabilities during their development,albeit through various mechanisms. Such capabilities include evadingapoptosis, self-sufficiency in growth signals, insensitivity toanti-growth signals, tissue invasion/metastasis, limitless replicativepotential, and sustained angiogenesis. The term “cancer cell” is meantto encompass both pre-malignant and malignant cancer cells.

As used herein, the phrase “cancer cell phenotype inhibiting” refers tothe ability of a compound to prevent or reduce cancer cell colonyformation in soft agar or tubular network formation in athree-dimensional basement membrane or extracellular matrix preparationor any other method that detects a reduction in a cancer cell phenotype,for example, assays that detect an increase in contact inhibition ofcell proliferation (e.g., reduction of colony formation in a monolayercell culture). Cancer cell phenotype inhibiting compounds may also causea reduction or elimination of colonies when added to establishedcolonies of cancer cells in soft agar or the extent of tubular networkformation in a three-dimensional basement membrane or extracellularmatrix preparation. EphA4 antibodies that inhibit cancer cell phenotypemay or may not also agonize EphA4 and may or may not have a low K_(off)rate.

The term “derivative” as used herein refers to a polypeptide thatcomprises an amino acid sequence of an EphA4 polypeptide, a fragment ofan EphA4 polypeptide, an antibody that immunospecifically binds to anEphA4 polypeptide, or an antibody fragment that immunospecifically bindsto an EphA4 polypeptide, that has been altered by the introduction ofamino acid residue substitutions, deletions or additions. The term“derivative” as used herein also refers to an EphA4 polypeptide, afragment of an EphA4 polypeptide, an antibody that immunospecificallybinds to an EphA4 polypeptide, or an antibody fragment thatimmunospecifically binds to an EphA4 polypeptide which has beenmodified, i.e, by the covalent attachment of any type of molecule to thepolypeptide. For example, but not by way of limitation, an EphA4polypeptide, a fragment of an EphA4 polypeptide, an antibody, orantibody fragment may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative of an EphA4 polypeptide, afragment of an EphA4 polypeptide, an antibody, or antibody fragment maybe modified by chemical modifications using techniques known to those ofskill in the art, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative of an EphA4 polypeptide, a fragment of anEphA4 polypeptide, an antibody, or antibody fragment may contain one ormore non-classical amino acids. In one embodiment, a polypeptidederivative possesses a similar or identical function as an EphA4polypeptide, a fragment of an EphA4 polypeptide, an antibody, orantibody fragment described herein. In another embodiment, a derivativeof EphA4 polypeptide, a fragment of an EphA4 polypeptide, an antibody,or antibody fragment has an altered activity when compared to anunaltered polypeptide. For example, a derivative antibody or fragmentthereof can bind to its epitope more tightly or be more resistant toproteolysis.

The term “epitope” as used herein refers to a portion of an EphA4polypeptide having antigenic or immunogenic activity in an animal,preferably in a mammal, and most preferably in a mouse or a human. Anepitope having immunogenic activity is a portion of an EphA4 polypeptidethat elicits an antibody response in an animal. An epitope havingantigenic activity is a portion of an EphA4 polypeptide to which anantibody immunospecifically binds as determined by any method well knownin the art, for example, by immunoassays. Antigenic epitopes need notnecessarily be immunogenic.

The “fragments” described herein include a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anEphA4 polypeptide or an antibody that immunospecifically binds to anEphA4 polypeptide. Preferably, antibody fragments are epitope-bindingfragments.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g., murine) antibodies that are chimeric antibodies whichcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and capacity. In some instances,Framework Region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues which are not found in the recipient antibody orin the donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody is aderivative. Such a humanized antibody comprises amino acid residuesubstitutions, deletions or additions in one or more non-human CDRs. Thehumanized antibody derivative may have substantially the same binding,better binding, or worse binding when compared to a non-derivativehumanized antibody. In specific embodiments, one, two, three, four, orfive amino acid residues of the CDR have been substituted, deleted oradded (i.e., mutated). For further details in humanizing antibodies, seeEuropean Patent Nos. EP 239,400, EP 592,106, and EP 519,596;International Publication Nos. WO 91/09967 and WO 93/17105; U.S. Pat.Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and6,407,213; and Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska etal., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-25;Caldas et al., 2000, Protein Eng. 13:353-60; Morea et al., 2000, Methods20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-84; Roguska etal., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55(23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22;Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol.235:959-73; Jones et al., 1986, Nature 321:522-525; Reichmann et al.,1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol.2:593-596.

As used herein, the term “hyperproliferative cell disorder” refers to adisorder that is not neo-plastic in which cellular hyperproliferationcauses or contributes to the pathological state or symptoms of thedisorder. In some embodiments, the hyperproliferative cell disorder ischaracterized by hyperproliferating epithelial cells. Hyperproliferativeepithelial cell disorders include, but are not limited to, asthma, COPD,lung fibrosis, bronchial hyper responsiveness, psoriasis, seborrheicdermatitis, and cystic fibrosis. In other embodiments, thehyperproliferative cell disorder is characterized by hyperproliferatingendothelial cells. Hyperproliferative endothelial cell disordersinclude, but are not limited to restenosis, hyperproliferative vasculardisease, Behcet's Syndrome, atherosclerosis, and macular degeneration.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen binding. Thehypervariable region comprises amino acid residues from a“Complementarity Determining Region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with ahyperproliferative cell disorder, especially cancer. A firstprophylactic or therapeutic agent can be administered prior to (e.g., 1minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second prophylactic or therapeutic agent to asubject which had, has, or is susceptible to a hyperproliferative celldisorder, especially cancer. The prophylactic or therapeutic agents areadministered to a subject in a sequence and within a time interval suchthat the agent of the invention can act together with the other agent toprovide an increased benefit than if they were administered otherwise.Any additional prophylactic or therapeutic agent can be administered inany order with the other additional prophylactic or therapeutic agents.

As used herein, the phrase “low tolerance” refers to a state in whichthe patient suffers from side effects from treatment so that the patientdoes not benefit from and/or will not continue therapy because of theadverse effects and/or the harm from the side effects outweighs thebenefit of the treatment.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from administration of aprophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore prophylactic or therapeutic agents to “manage” a disease so as toprevent the progression or worsening of the disease.

As used herein, the term “neoplastic” refers to a disease involvingcells that have the potential to metastasize to distal sites and exhibitphenotypic traits that differ from those of non-neoplastic cells, forexample, formation of colonies in a three-dimensional substrate such assoft agar or the formation of tubular networks or weblike matrices in athree-dimensional basement membrane or extracellular matrix preparation,such as MATRIGEL™. Non-neoplastic cells do not form colonies in softagar and form distinct sphere-like structures in three-dimensionalbasement membrane or extracellular matrix preparations. Neoplastic cellsacquire a characteristic set of functional capabilities during theirdevelopment, albeit through various mechanisms. Such capabilitiesinclude evading apoptosis, self-sufficiency in growth signals,insensitivity to anti-growth signals, tissue invasion/metastasis,limitless replicative potential, and sustained angiogenesis. Thus,“non-neoplastic” means that the condition, disease, or disorder does notinvolve cancer cells.

As used herein, the phrase “non-responsive/refractory” is used todescribe patients treated with one or more currently available therapies(e.g., cancer therapies) such as chemotherapy, radiation therapy,surgery, hormonal therapy and/or biological therapy/immunotherapy,particularly a standard therapeutic regimen for the particular cancer,wherein the therapy is not clinically adequate to treat the patientssuch that these patients need additional effective therapy, e.g., remainunsusceptible to therapy. The phrase can also describe patients whorespond to therapy yet suffer from side effects, relapse, developresistance, etc. In various embodiments, “non-responsive/refractory”means that at least some significant portion of the cancer cells are notkilled or their cell division arrested. The determination of whether thecancer cells are “non-responsive/refractory” can be made either in vivoor in vitro by any method known in the art for assaying theeffectiveness of treatment on cancer cells, using the art-acceptedmeanings of “refractory” in such a context. In various embodiments, acancer is “non-responsive/refractory” where the number of cancer cellshas not been significantly reduced, or has increased during thetreatment.

As used herein, the term “potentiate” refers to an improvement in theefficacy of a therapeutic agent at its common or approved dose.

As used herein, the terms “prevent,” “preventing” and “prevention” referto the prevention of the onset, recurrence, or spread of a disease in asubject resulting from the administration of a prophylactic ortherapeutic agent.

As used herein, the term “prophylactic agent” refers to any agent thatcan be used in the prevention of the onset, recurrence or spread of adisease or disorder associated with EphA4 overexpression and/or cellhyperproliferative disease, particularly cancer. In certain embodiments,the term “prophylactic agent” refers to an EphA4 agonistic antibody, anEphA4 cancer cell phenotype inhibiting antibody, an exposed EphA4epitope antibody, or an antibody that binds EphA4 with a low K_(off). Incertain other embodiments, the term “prophylactic agent” refers tocancer chemotherapeutics, radiation therapy, hormonal therapy,biological therapy (e.g., immunotherapy), and/or EphA4 antibodies of theinvention. In other embodiments, more than one prophylactic agent may beadministered in combination.

As used herein, a “prophylactically effective amount” refers to thatamount of the prophylactic agent sufficient to result in the preventionof the onset, recurrence or spread of cell hyperproliferative disease,preferably, cancer. A prophylactically effective amount may refer to theamount of prophylactic agent sufficient to prevent the onset, recurrenceor spread of hyperproliferative disease, particularly cancer, includingbut not limited to those predisposed to hyperproliferative disease, forexample, those genetically predisposed to cancer or previously exposedto carcinogens. A prophylactically effective amount may also refer tothe amount of the prophylactic agent that provides a prophylacticbenefit in the prevention of hyperproliferative disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of hyperproliferative disease. Used in connection with anamount of an EphA4 antibody of the invention, the term can encompass anamount that improves overall prophylaxis or enhances the prophylacticefficacy of or synergies with another prophylactic agent.

As used herein, a “protocol” includes dosing schedules and dosingregimens.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Adverse effectsare always unwanted, but unwanted effects are not necessarily adverse.An adverse effect from a prophylactic or therapeutic agent might beharmful or uncomfortable or risky. Side effects from chemotherapyinclude, but are not limited to, gastrointestinal toxicity such as, butnot limited to, early and late-forming diarrhea and flatulence, nausea,vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure,as well as constipation, nerve and muscle effects, temporary orpermanent damage to kidneys and bladder, flu-like symptoms, fluidretention, and temporary or permanent infertility. Side effects fromradiation therapy include but are not limited to fatigue, dry mouth, andloss of appetite. Side effects from biological therapies/immunotherapiesinclude but are not limited to rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills and fatigue,digestive tract problems and allergic reactions. Side effects fromhormonal therapies include but are not limited to nausea, fertilityproblems, depression, loss of appetite, eye problems, headache, andweight fluctuation. Additional undesired effects typically experiencedby patients are numerous and known in the art. Many are described in thePhysicians' Desk Reference (56^(th) ed., 2002).

As used herein, the terms “single-chain Fv” and “scFv” refer to antibodyfragments comprising the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of scFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). In specific embodiments,scFvs include bi-specific scFvs and humanized scFvs.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

As used herein, the terms “treat,” “treating” and “treatment” refer tothe eradication, reduction or amelioration of symptoms of a disease ordisorder, particularly, the eradication, removal, modification, orcontrol of primary, regional, or metastatic cancer tissue that resultsfrom the administration of one or more therapeutic agents. In certainembodiments, such terms refer to the minimizing or delaying the spreadof cancer resulting from the administration of one or more therapeuticagents to a subject with such a disease.

As used herein, the term “therapeutic agent” refers to any agent thatcan be used in the prevention, treatment, or management of a disease ordisorder associated with overexpression of EphA4 and/or cellhyperproliferative diseases or disorders, particularly, cancer. Incertain embodiments, the term “therapeutic agent” refers to an EphA4agonistic antibody, an EphA4 cancer cell phenotype inhibiting antibody,an exposed EphA4 epitope antibody, or an antibody that binds EphA4 witha K_(off) of less than 3×10⁻³ s⁻¹, a non-antibody agent that agonizesEphA4, such as an EphA4 ligand or EphA4-binding fragment or derivativethereof, or an agent that reduces EphA4 expression, such as EphA4 RNAi,antisense, etc. In certain other embodiments, the term “therapeuticagent” refers to cancer chemotherapeutics, radiation therapy, hormonaltherapy, biological therapy/immunotherapy, and/or EphA4 antibody of theinvention. In other embodiments, more than one therapeutic agent may beadministered in combination.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder associated with EphA4 overexpression and/or cellhyperproliferative disease and, preferably, the amount sufficient todestroy, modify, control or remove primary, regional or metastaticcancer tissue. A therapeutically effective amount may refer to theamount of therapeutic agent sufficient to delay or minimize the onset ofthe hyperproliferative disease, e.g., delay or minimize the spread ofcancer. A therapeutically effective amount may also refer to the amountof the therapeutic agent that provides a therapeutic benefit in thetreatment or management of cancer. Further, a therapeutically effectiveamount with respect to a therapeutic agent of the invention means thatamount of therapeutic agent alone, or in combination with othertherapies, that provides a therapeutic benefit in the treatment ormanagement of hyperproliferative disease or cancer. Used in connectionwith an amount of an EphA4 antibody of the invention, the term canencompass an amount that improves overall therapy, reduces or avoidsunwanted effects, or enhances the therapeutic efficacy of or synergieswith another therapeutic agent.

4. DESCRIPTION OF THE FIGURES

FIG. 1: EphA4 antibodies induce cell rounding. Aspc1 cells incubatedwith single chain Fv antibodies (scFv) that bind EphA4 show cellrounding when incubated at 37° C. relative to cells incubated at 0° C.

FIG. 2: Crosslinking of EphA4 causes tyrosine phosphorylation of anassociated 75 kDa protein. Cells were incubated with LX13 scFv, thencrosslinked by anti-Flag antibody, immunoprecipitated with Ephrin A4-Fc,and blotted with anti-phosphotyrosine. Cross-linked EphA4 (“X-linkedEphA4”) shows increased tyrosine phosphorylation of an EphA4-associated75 kDa protein, relative to control cells (“Control”) that are notcross-linked.

FIG. 3: EphA4 aggregation inhibits soft agar growth of ASPC1 cells.Cells treated with EphA4 antibodies (scFv), which are then cross-linkedwith a secondary monoclonal antibody (mab; “LX13 Bival”), showsignificant decrease in colony formation in soft agar relative to cellstreated with scFv alone (“LX13”) or secondary mab alone (“Control”).

FIG. 4: EphA4 upregulation causes a decreased dependence on estrogen forcell growth in a mammary carcinoma cell line. MCF-7 cells, whichnormally express very low levels of EphA4, were transfected with emptyvector (“Vector”) or vector containing EphA4 sequence. Stable expressorsof EphA4 were isolated (clones 2 and 3, “EphA4-2” and “EphA4-3”). Insoft agar assays in which the cells were incubated with estrogen,MCF-7-EphA4 cells demonstrated a 2.4 fold increase in anchorageindependent growth compared to a vector-transfected control (“+Estrogen”). However in the absence of estrogen (“− Estrogen”),EphA4-expressing cells had an 8.5 fold increase in growth compared tovector-transfected controls.

FIG. 5: EphA4 RNA levels are increased in pancreatic tumor tissue. BarA: EphA4 RNA in pathologically normal pancreatic tissue from 52-year oldmale. Bar B: EphA4 RNA in pancreatic tissue displaying Stage 4A ductalpancreatic adenocarcinoma from same 52-year old male as in Bar A. Bar C:EphA4 RNA in pancreatic tissue displaying Stage 2A ductal pancreaticadenocarcinoma from 72-year old male. Bar D: EphA4 RNA in lymph nodetissue of 71-year old female displaying Stage 2B metastatic pancreaticadenocarcinoma. Bar E: EphA4 RNA in omentum tissue of 57-year old femaledisplaying Stage 4 metastatic pancreatic adenocarcinoma. Bar F: EphA4RNA in liver tissue of 45-year old female displaying metastaticpancreatic adenocarcinoma.

FIG. 6: Anti-EphA4 scFv clone 44 strongly induces tyrosinephosphorylation of EphA4. Anti-EphA4 scFv clones 8, 18, and 20 alsoweakly induce EphA4 tyrosine phosphorylation, relative to control cellsin media or cells incubated with anti-Flag antibodies alone, which showlow levels of EphA4 phosphorylation.

FIG. 7: Sequence of scFV clone EphA4-44. CDR, VH, and VL domains areindicated.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the inventors' discoverythat EphA4 is upregulated in certain cancer cells and anti-EphA4antibodies reduce certain cancer cell behaviors, such as cell attachmentand growth in soft agar. Anti-EphA4 antibodies promote phosphorylationof EphA4 and an EphA4-associated protein. The present inventors alsobelieve that EphA4 plays a role in cancer and cell proliferation similarto EphA2. EphA2 monoclonal antibodies that agonize EphA2 can inhibitcancer cell proliferation and invasiveness by reducing the levels ofEphA2 expression in these cancer cells (see co-pending U.S. patentapplication Ser. No. 10/436,782, entitled “EphA2 Monoclonal Antibodiesand Methods of Use Thereof” filed May 12, 2003). Decreased EphA4activity or other results of EphA4 signaling may selectively inhibitmalignant cancer cell growth. In particular, decreased levels of EphA4and phosphorylation of EphA4 and/or a 75 kDa EphA4-associated proteinmay be achieved with EphA4 agonistic monoclonal antibodies. Although notintending to be bound by any mechanism of action, this inhibition ofcell growth and/or metastasis may be achieved by stimulating (i.e.,agonizing) EphA4 signaling thereby causing EphA4 phosphorylation whichleads to the degradation of EphA4. Cancer cell growth may be decreaseddue to the decreased EphA4 levels and, therefore, decreasedligand-independent EphA4 signaling. Decreased EphA4 activity may also beachieved with EphA4 cancer cell phenotype inhibiting antibodies orantibodies that preferentially bind an EphA4 epitope exposed on cancercells but not non-cancer cells. Additionally, antibodies that bind EphA4with a low K_(off) (e.g., less than 3×10⁻³ s⁻¹) may also decrease EphA4levels.

In addition, the inventors have found that antibody binding to EphA4 issufficient to trigger changes in cell behavior. Specifically, EphA4antibody binding to cells expressing EphA4 decreases cell-ECMattachments and induces cell rounding. Cell-ECM attachments aregenerally understood to provide physical attachments and intracellularsignaling that govern many aspects of cell behavior, including decisionsregarding cell growth, migration, invasion and differentiation.Consistent with this understanding, changes in cell interaction with itsmicroenvironment have been linked with the initiation or progression ofmany different disease states. For example, increased ECM attachment canpromote migration and proliferation and thereby triggerhyperproliferative diseases, which include but are not limited to:cancer; inflammatory diseases of the bowel (IBD), intestine (Crohn'sdisease), stomach and other vital organs; asthma; COPD and otherhyperproliferative diseases of the lung; and restenosis, which is aclinical manifestation resulting from increased growth and migration ofsmooth muscle or endothelial cells. Therefore, EphA4 antibodies can beuseful in the treatment of non-cancerous conditions in general, and fortreatment of conditions linked with cell-ECM attachments in particular.

Accordingly, the present invention relates to methods and compositionsthat provide for the treatment, inhibition, and management of diseasesand disorders associated with overexpression of EphA4 and/or cellhyperproliferative diseases and disorders. A particular aspect of theinvention relates to methods and compositions containing compounds thatinhibit cancer cell proliferation and invasion, particularly thosecancer cells that overexpress EphA4. The present invention furtherrelates to methods and compositions for the treatment, inhibition, ormanagement of metastases of cancers of epithelial cell origin,especially human cancers of the breast, lung, skin, prostate, bladder,and pancreas, and renal cell carcinomas and melanomas. Furthercompositions and methods of the invention include other types of activeingredients in combination with the EphA4 antibodies of the invention.In other embodiments, the methods of the invention are used to treat,prevent or manage other diseases or disorders associated with cellhyperproliferation, for example but not limited to asthma, psoriasis,restenosis, COPD, etc.

The present invention also relates to methods for the treatment,inhibition, and management of cancer or other hyperproliferative celldisorder or disease that has become partially or completely refractoryto current or standard cancer treatment, such as chemotherapy, radiationtherapy, hormonal therapy, and biological therapy.

The invention further provides diagnostic methods using the EphA4antibodies of the invention, particularly the exposed EphA4 epitopeantibodies, to evaluate the efficacy of cancer treatment, eitherEphA4-based or not EphA4-based. The diagnostic methods of the inventioncan also be used to prognose or predict cancer progression. Inparticular embodiments, the diagnostic methods of the invention providemethods of imaging and localizing metastases and methods of diagnosisand prognosis using tissues and fluids distal to the primary tumor site(as well as methods using tissues and fluids of the primary tumor). Inother embodiments, the diagnostic methods of the invention providemethods of imaging and localizing metastases and methods of diagnosisand prognosis in vivo.

In an additional embodiment, the invention provides methods of screeningfor anti-cancer agents, particularly anti-metastatic cancer agents, byscreening agents for the ability to decrease cell colonization in softagar and/or tubular network formation in three-dimensional basementmembrane and extracellular matrix preparations, such as MATRIGEL™. Inpreferred embodiments, the invention provides methods of screening foragents for the treatment and prevention of hyperproliferative diseasesand disorders by assaying for the ability to reduce the extent ofexisting cell colonization in soft agar and/or tubular network formationin three-dimensional basement membrane. The present inventors found thatinhibition of cell colonization in soft agar and/or tubular networkformation in MATRIGEL™ is a far better indication of antimetastaticactivity and may identify potential anti-metastatic agents that wouldnot have been identified by standard cell culture assays.

5.1 Antibodies

As discussed above, the invention encompasses administration ofantibodies (preferably monoclonal antibodies) or fragments thereof thatimmunospecifically bind to and agonize EphA4 signaling (“EphA4 agonisticantibodies”); inhibit a cancer cell phenotype, e.g., inhibit colonyformation in soft agar or tubular network formation in athree-dimensional basement membrane or extracellular matrix preparation,such as MATRIGEL™ (“cancer cell phenotype inhibiting antibodies”);preferentially bind epitopes on EphA4 that are selectively exposed orincreased on cancer cells but not non-cancer cells (“exposed EphA4epitope antibodies”); and/or bind EphA4 with a K_(off) of less than3×10⁻³ s⁻¹. In one embodiment, the antibody binds to the extracellulardomain of EphA4 and, preferably, also agonizes EphA4, e.g., increasesEphA4 phosphorylation and/or phosphorylation of the 75 kDaEphA4-associated protein and, preferably, causes EphA4 degradation. Inanother embodiment, the antibody binds to the extracellular domain ofEphA4 and, preferably, also inhibits and, even more preferably, reducesthe extent of (e.g., by cell killing mechanisms such as necrosis andapoptosis) colony formation in soft agar or tubular network formation ina three-dimensional basement membrane or extracellular matrixpreparation. In other embodiments, the antibodies inhibit or reduce acancer cell phenotype in the presence of another anti-cancer agent, suchas a hormonal, biologic, chemotherapeutic or other agent. In anotherembodiment, the antibody binds to the extracellular domain of EphA4 atan epitope that is exposed in a cancer cell but occluded in a non-cancercell. In another embodiment, the antibody binds to the extracellulardomain of EphA4, preferably with a K_(off) of less than 1×10⁻³ s⁻¹, morepreferably less than 3×10⁻³ s⁻¹. In other embodiments, the antibodybinds to EphA4 with a K_(off) of less than 10⁻³ s⁻¹, less than 5×10⁻³s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, lessthan 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In a more preferred embodiment, the antibody binds to an EphA4 epitopeand/or competes for EphA4 binding as assayed by ELISA or any otherappropriate immunoassay. Cells that express the anti-EphA4 scFv EA44have been deposited with the American Type Culture Collection (P.O. Box1549, Manassas, Va. 20108) on Jun. 1, 2004 under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedures, and assignedaccession number _______ and is hereby incorporated by reference. In aspecific embodiment, the invention provides the EphA4 scFv antibodyEphA4-44/EA44 (in the specification herein, EA44 and EphA4-44 refer tothe same antibody, that is, the EphA4-44 scFv clone which binds to EphA4and was deposited with the ATCC on Jun. 10, 2004 as “EA44”). In apreferred embodiment, the invention provides an EphA4 antibody whereinthe variable heavy chain and/or variable light chain amino acid sequencepossesses at least 90% sequence identity with the variable heavy chainor light chain amino acid sequence of EA44, as contained SEQ IDs NO: 4and 8, respectively. In other preferred embodiments, the inventionprovides an EphA4 antibody wherein at least three, at least four, atleast five, or all six of its CDRs are identical to the correspondingCDRs in EA44. In other specific embodiments, the invention providesanti-EphA4 scFv clones 8, 18, 20, 36, and 41. In a most preferredembodiment, the antibody is human or has been humanized.

In other embodiments, the EphA4 antibody comprises the EA44 VH CDR1 (SEQID NO. 22) and VL CDR1 (SEQ ID NO. 28); the EA44 VH CDR1 (SEQ ID NO. 22)and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22) and VLCDR3 (SEQ ID NO. 32); the EA44 VH CDR2 (SEQ ID NO. 24) and VL CDR1 (SEQID NO. 28); the EA44 VH CDR2 (SEQ ID NO. 24) and VL CDR2 (SEQ ID NO.30); the EA44 VH CDR2 (SEQ ID NO. 24) and VH CDR3 (SEQ ID NO. 26); theEA44 VH CDR3 (SEQ ID NO. 26) and VH CDR1 (SEQ ID NO. 22); the EA44 VHCDR3 (SEQ ID NO. 26) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR3 (SEQID NO. 26) and VL CDR3 (SEQ ID NO. 32); the EA44 VH1 CDR1, VH CDR2 (SEQID NO. 24) and VL CDR1 (SEQ ID NO. 28); the EA44 VH CDR1 (SEQ ID NO.22), VH CDR2 (SEQ ID NO. 24) and VL CDR2 (SEQ ID NO. 30); the EA44 VHCDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24) and VL CDR3 (SEQ ID NO.32); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26) and VLCDR1 (SEQ ID NO. 28), the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ IDNO. 26) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR2 (SEQ ID NO. 24),VH CDR2 (SEQ ID NO. 24) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1(SEQ ID NO. 22), VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30);the EA44 VH CDR1 (SEQ ID NO. 22), VL CDR1 (SEQ ID NO. 28) and VL CDR3(SEQ ID NO. 32); the EA44 VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO.28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR2 (SEQ ID NO. 24), VLCDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR3 (SEQID NO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30); theEA44 VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQID NO. 32); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24),VH CDR3 (SEQ ID NO. 26) and VL CDR1 (SEQ ID NO. 28); the EA44 VH CDR1(SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26) and VLCDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ IDNO. 24), VH CDR3 (SEQ ID NO. 26) and VL CDR3 (SEQ ID NO. 32); the EA44VHCDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28)and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR2(SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32);the EA44 VH CDR1 (SEQ ID NO. 22), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR1 (SEQ ID NO.22), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQID NO. 32); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26),VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VH CDR2(SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28) and VLCDR3 (SEQ ID NO. 32); the EA44 VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ IDNO. 26), VL CDR2 (SEQ ID NO. 30) and VL CDR3 (SEQ ID NO. 32); the EA44VH CDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO.26), VL CDR1 (SEQ ID NO. 28) and VL CDR2 (SEQ ID NO. 30); the EA44 VHCDR1 (SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VH CDR3 (SEQ ID NO. 26),VL CDR1 (SEQ ID NO. 28) and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1(SEQ ID NO. 22), VH CDR2 (SEQ ID NO. 24), VL CDR1 (SEQ ID NO. 28), VLCDR2 (SEQ ID NO. 30), and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR1 (SEQID NO. 22), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28), VL CDR2(SEQ ID NO. 30), and VL CDR3 (SEQ ID NO. 32); the EA44 VH CDR2 (SEQ IDNO. 24), VH CDR3 (SEQ ID NO. 26), VL CDR1 (SEQ ID NO. 28), VL CDR2 (SEQID NO. 30), and VL CDR3 (SEQ ID NO. 32); or any combination thereof ofthe EA44 VH CDRs and VL CDRs listed in FIG. 7.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, synthetic antibodies, recombinantly produced antibodies,intrabodies, multispecific antibodies (including bi-specificantibodies), human antibodies, humanized antibodies, chimericantibodies, single-chain Fvs (scFv) (including bi-specific scFvs) suchas scFv clones 8, 18, 20, 36, 41, and EA44, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and epitope-binding fragments ofany of the above. In particular, antibodies used in the methods of thepresent invention include immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds to EphA4and is an agonist of EphA4, inhibits or reduces a cancer cell phenotype,preferentially binds an EphA4 epitope exposed on cancer cells but notnon-cancer cells, and/or binds EphA4 with a K_(off) of less than 3×10⁻³s⁻¹. The immunoglobulin molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule.

The antibodies used in the methods of the invention may be from anyanimal origin including birds and mammals (e.g., human, murine, donkey,sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably,the antibodies are human or humanized monoclonal antibodies. As usedherein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from mice or other animals thatexpress antibodies from human genes.

The antibodies used in the methods of the present invention may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may immunospecifically bind to differentepitopes of an EphA4 polypeptide or may immunospecifically bind to bothan EphA4 polypeptide as well a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g.,International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360,and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat.Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; andKostelny et al., 1992, J. Immunol. 148:1547-1553.

In a preferred embodiment, antibodies of the invention are bispecific Tcell engagers (BiTEs). Bispecific T cell engagers (BiTE) are bispecificantibodies that can redirect T cells for antigen-specific elimination oftargets. A BiTE molecule has an antigen-binding domain that binds to a Tcell antigen (e.g. CD3) at one end of the molecule and an antigenbinding domain that will bind to an antigen on the target cell. A BiTEmolecule was recently described in WO 99/54440, which is hereinincorporated by reference. This publication describes a novelsingle-chain multifunctional polypeptide that comprises binding sitesfor the CD19 and CD3 antigens (CD19×CD3). This molecule was derived fromtwo antibodies, one that binds to CD19 on the B cell and an antibodythat binds to CD3 on the T cells. The variable regions of thesedifferent antibodies are linked by a polypeptide sequence, thus creatinga single molecule. Also described, is the linking of the variable heavychain (VH) and light chain (VL) of a specific binding domain with aflexible linker to create a single chain, bispecific antibody.

In an embodiment of this invention, an antibody or ligand thatimmunospecifically binds to EphA4 will comprise a portion of the BiTEmolecule. For example, the VH and/or VL (preferably a scFV) of anantibody that binds EphA4 can be fused to an anti-CD3 binding portionsuch as that of the molecule described above, thus creating a BiTEmolecule that targets EphA4. In addition to the variable heavy and orlight chain of antibody against EphA4, other molecules that bind EphA4can comprise the BiTE molecule, for example Ephrins A1, A2, A3, A4, A5,B2, and B3; B61, AL1/RAGS, LERK4, Htk-L, and Elk-L3. In anotherembodiment, the BiTE molecule can comprise a molecule that binds toother T cell antigens (other than CD3). For example, ligands and/orantibodies that immunospecifically bind to T-cell antigens like CD2,CD4, CD8, CD11a, TCR, and CD28 are contemplated to be part of thisinvention. This list is not meant to be exhaustive but only toillustrate that other molecules that can immunospecifically bind to a Tcell antigen can be used as part of a BiTE molecule. These molecules caninclude the VH and/or VL portions of the antibody or natural ligands(for example LFA3 whose natural ligand is CD3). In one embodiment of theinvention, the BiTE molecule is an EphA4 agonist. In another embodiment,the BiTE molecule is an EphA4 antagonist.

The “binding domain” as used in accordance with the present inventiondenotes a domain comprising a three-dimensional structure capable ofspecifically binding to an epitope like native antibodies, free scFvfragments or one of their corresponding immunoglobulin chains,preferably the VH chain. Thus, said domain can comprise the VH and/or VLdomain of an antibody or an immunoglobulin chain, preferably at leastthe VH domain or more preferably the VH and VL domain linked by aflexible polypeptide linker (scFv). On the other hand, said bindingdomain contained in the polypeptide of the invention may comprise atleast one complementarity determining region (CDR) of an antibody orimmunoglobulin chain recognizing an antigen on the T cell or a cellularantigen. In this respect, it is noted that the binding domain present inthe polypeptide of the invention may not only be derived from antibodiesbut also from other T cell or cellular antigen binding protein, such asnaturally occurring surface receptors or ligands. It is furthercontemplated that in an embodiment of the invention, said first and orsecond domain of the above-described polypeptide mimic or correspond toa VH and VL region from a natural antibody. The antibody providing thebinding site for the polypeptide of the invention can be, e.g., amonoclonal antibody, polyclonal antibody, chimeric antibody, humanizedantibody, bispecific antibody, synthetic antibody, antibody fragment,such as Fab, Fv or scFv fragments etc., or a chemically modifiedderivative of any of these.

The antibodies used in the methods of the invention include derivativesthat are modified, i.e, by the covalent attachment of any type ofmolecule to the antibody. For example, but not by way of limitation, theantibody derivatives include antibodies that have been modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The present invention encompasses single domain antibodies, includingcamelized single domain antibodies (see e.g., Muyldermans et al., 2001,Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech.1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25;International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Pat.No. 6,005,079; which are incorporated herein by reference in theirentireties). In one embodiment, the present invention provides singledomain antibodies comprising two VH domains having the amino acidsequence of any of the VH domains of an EphA4 agonistic antibody, EphA4cancer cell phenotype inhibiting antibody, exposed EphA4 epitopeantibody, or an EphA4 antibody that binds EphA4 with a K_(off) of lessthan 3×10⁻³ s⁻¹ with modifications such that single domain antibodiesare formed. In another embodiment, the present invention also providessingle domain antibodies comprising two VH domains comprising one ormore of the VH CDRs of an EphA4 agonistic antibody, EphA4 cancer cellphenotype inhibiting antibody, exposed EphA4 epitope antibody, or anEphA4 antibody that binds EphA4 with a K_(off) of less than 3×10⁻³ s⁻¹.

The methods of the present invention also encompass the use ofantibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the antibodies of thepresent invention or fragments thereof in a mammal, preferably a human,result in a higher serum titer of said antibodies or antibody fragmentsin the mammal, and thus, reduce the frequency of the administration ofsaid antibodies or antibody fragments and/or reduces the concentrationof said antibodies or antibody fragments to be administered. Antibodiesor fragments thereof having increased in vivo half-lives can begenerated by techniques known to those of skill in the art. For example,antibodies or fragments thereof with increased in vivo half-lives can begenerated by modifying (e.g., substituting, deleting or adding) aminoacid residues identified as involved in the interaction between the Fcdomain and the FcRn receptor (see, e.g., International Publication Nos.WO 97/34631 and WO 02/060919, which are incorporated herein by referencein their entireties). Antibodies or fragments thereof with increased invivo half-lives can be generated by attaching to said antibodies orantibody fragments polymer molecules such as high molecular weightpolyethyleneglycol (PEG). PEG can be attached to said antibodies orantibody fragments with or without a multifunctional linker eitherthrough site-specific conjugation of the PEG to the N- or C-terminus ofsaid antibodies or antibody fragments or via epsilon-amino groupspresent on lysine residues. Linear or branched polymer derivatizationthat results in minimal loss of biological activity will be used. Thedegree of conjugation will be closely monitored by SDS-PAGE and massspectrometry to ensure proper conjugation of PEG molecules to theantibodies. Unreacted PEG can be separated from antibody-PEG conjugatesby, e.g., size exclusion or ion-exchange chromatography.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

The present invention further encompasses antibodies or fragmentsthereof that immunospecifically bind to EphA4 and agonize EphA4 and/orinhibit a cancer cell phenotype, preferentially bind an EphA4 epitopeexposed in cancer cells, and/or bind EphA4 with a K_(off) of less than3×10⁻³ s⁻¹, said antibodies or antibody fragments comprising an aminoacid sequence of one or more CDRs that is at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of one or more CDRs of an EphA4antibody. The determination of percent identity of two amino acidsequences can be determined by any method known to one skilled in theart, including BLAST protein searches.

In a specific embodiment, the present invention encompasses antibodiesor antibody fragments comprising an amino acid sequence of one or moreCDRs that is at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical to the amino acidsequence of one or more CDRs of scFv clone EA44. In a furtherembodiment, the invention encompasses a recombinant EphA4 antibody orantibody fragment that is an EphA4 agonistic antibody, an EphA4 cancercell phenotype inhibiting antibody, or an exposed EphA4 epitope antibodywherein the antibody comprises at least one of the complementaritydetermining regions (CDRs) of the variable light chain or variable heavychain of the EA44 antibody wherein the amino acid sequences of thevariable heavy and light chain sequences of EA44.

In another specific embodiment, the invention encompasses an EphA4antibody or antibody fragment that is an EphA4 agonistic antibody, anEphA4 cancer cell phenotype inhibiting antibody, or an exposed EphA4epitope antibody wherein said antibody comprises one, two, three, four,five, or all six of the complementarity determining regions (CDRs) ofthe variable light chain and/or variable heavy chain of the EA44antibody.

5.1.1 Antibody Conjugates

The present invention encompasses the use of antibodies or fragmentsthereof (or other therapeutics, such as EphA4 ligands and EphA4-bindingfragments thereof) recombinantly fused or chemically conjugated(including both covalent and non-covalent conjugations) to aheterologous agent. The heterologous agent may be a polypeptide (orportion thereof, preferably to a polypeptide of at least 10, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90 or at least 100 amino acids), nucleic acid, smallmolecule (less than 1000 daltons), or inorganic or organic compound. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. Antibodies fused or conjugated to heterologous agentsmay be used in vivo to detect, treat, manage, or monitor the progressionof a disorder using methods known in the art. See e.g., InternationalPublication WO 93/21232; EP 439,095; Naramura et al., 1994, Immunol.Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452, whichare incorporated by reference in their entireties. In some embodiments,the disorder to be detected, treated, managed, or monitored is malignantcancer that overexpresses EphA4. In other embodiments, the disorder tobe detected, treated, managed, or monitored is pre-malignant cancer thatoverexpresses EphA4. In a specific embodiments, the pre-malignant canceris high-grade prostatic intraepithelial neoplasia (PIN), ductalcarcinoma of the breast, or compound nevi.

The present invention further includes compositions comprisingheterologous agents fused or conjugated to antibody fragments. Forexample, the heterologous polypeptides may be fused or conjugated to aFab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or portionthereof. Methods for fusing or conjugating polypeptides to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434;EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570;Ashkenazi et al., 1991, PNAS 88: 10535-10539; Meng et al., 1995, J.Immunol. 154:5590-5600; and ViI et al., 1992, PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16:76; Hansson, et al., 1999, J. Mol. Biol. 287:265; andLorenzo and Blasco, 1998, BioTechniques 24:308 (each of these patentsand publications are hereby incorporated by reference in its entirety).Antibodies or fragments thereof, or the encoded antibodies or fragmentsthereof, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding anantibody or antibody fragment, which portions immunospecifically bind toEphA4 may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous agents.

In one embodiment, antibodies of the present invention or fragments orvariants thereof are conjugated to a marker sequence, such as a peptide,to facilitate purification. In preferred embodiments, the marker aminoacid sequence is a hexa-histidine peptide, such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., 1989, PNAS 86:821, for instance, hexa-histidine providesfor convenient purification of the fusion protein. Other peptide tagsuseful for purification include, but are not limited to, thehemagglutinin “HA” tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) andthe “Flag” tag.

In other embodiments, antibodies of the present invention or fragmentsor variants thereof are conjugated to a diagnostic or detectable agent.Such antibodies can be useful for monitoring or prognosing thedevelopment or progression of a cancer as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Additionally, such antibodies can be useful for monitoring or prognosingthe development or progression of a pre-malignant cancer (e.g.,high-grade prostatic intraepithelial neoplasia (PIN), ductal carcinomaof the breast, or compound nevi). In one embodiment, an exposed EphA4epitope antibody is conjugated to a diagnostic or detectable agent.

Such diagnosis and detection can accomplished by coupling the antibodyto detectable substances including, but not limited to various enzymes,such as but not limited to horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such asbut not limited to streptavidin/biotin and avidin/biotin; fluorescentmaterials, such as but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to, bismuth (²¹³Bi), carbon (¹⁴C),chromium (⁵¹Cr), cobalt (⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium(¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I),lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum(⁹⁹Mo), palladium (¹⁰³Pd), phosphorous (³²P), praseodymium (¹⁴²Pr),promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), ruthemium(⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium(⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn,¹¹⁷Sn), tritium (³H), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium(⁹⁰Y), zinc (⁶⁵Zn); positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions.

In other embodiments, antibodies of the present invention or fragmentsor variants thereof are conjugated to a therapeutic agent such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, andcyclophosphamide and analogs or homologs thereof. Therapeutic agentsinclude, but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

In other embodiments, antibodies of the present invention or fragmentsor variants thereof are conjugated to a therapeutic agent or drug moietythat modifies a given biological response. Therapeutic agents or drugmoieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin, cholera toxin, or diphtheria toxin; a protein such as tumornecrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, anapoptotic agent, e.g., TNF-α, TNF-β, AIM I (see, InternationalPublication No. WO 97/33899), AIM II (see, International Publication No.WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Iminunol., 6:1567),and VEGI (see, International Publication No. WO 99/23105), a thromboticagent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or,a biological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

In other embodiments, antibodies of the present invention or fragmentsor variants thereof are conjugated to a therapeutic agent such as aradioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each incorporated by reference in their entireties.

In a specific embodiment, the conjugated antibody is an EphA4 antibodythat preferably binds an EphA4 epitope exposed on cancer cells but noton non-cancer cells (i.e., exposed EphA4 epitope antibody). In anotherspecific embodiment, the conjugated antibody is not EA2. In a furtherembodiment, the conjugated antibody comprises the variable light chainor variable heavy chain of the EA44 antibody.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58. Methods for fusing or conjugating antibodies topolypeptide moieties are known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP307,434; EP 367,166; International Publication Nos. WO 96/04388 and WO91/06570; Ashkenazi et al., 1991, PNAS 88: 10535-10539; Zheng et al.,1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, PNAS89:11337-11341. The fusion of an antibody to a moiety does notnecessarily need to be direct, but may occur through linker sequences.Such linker molecules are commonly known in the art and described inDenardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999,Bioconjug. Chem. 10:553; Zimmerman et al., 1999, Nucl. Med. Biol.26:943-50; Garnett, 2002, Adv. Drug Deliv. Rev. 53:171-216, each ofwhich is incorporated herein by reference in its entirety.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.1.2 Methods of Producing Antibodies

The antibodies or fragments thereof can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with EphA4 (either the full length protein or adomain thereof, e.g., the extracellular domain) and once an immuneresponse is detected, e.g., antibodies specific for EphA4 are detectedin the mouse serum, the mouse spleen is harvested and splenocytesisolated. The splenocytes are then fused by well known techniques to anysuitable myeloma cells, for example cells from cell line SP20 availablefrom the ATCC. Hybridomas are selected and cloned by limited dilution.Hybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, monoclonal antibodies can be generated by culturing ahybridoma cell secreting an antibody of the invention wherein,preferably, the hybridoma is generated by fusing splenocytes isolatedfrom a mouse immunized with EphA4 or fragment thereof with myeloma cellsand then screening the hybridomas resulting from the fusion forhybridoma clones that secrete an antibody able to bind EphA4.

Antibody fragments which recognize specific EphA4 epitopes may begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments of the invention may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the CH1 domain of the heavy chain. Further,the antibodies of the present invention can also be generated usingvarious phage display methods known in the art.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to the EphA4 epitope of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkman et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9; Burton et al., 1994, Advances in Immunology57:191-280; International Application No. PCT/GB91/01134; InternationalPublication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/11236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Phage may be screened for EphA4 binding, particularly to theextracellular domain of EphA4. Agonizing EphA4 activity (e.g.,increasing EphA4 phosphorylation, reducing EphA4 levels) or cancer cellphenotype inhibiting activity (e.g., reducing colony formation in softagar or tubular network formation in a three-dimensional basementmembrane or extracellular matrix preparation, such as MATRIGEL™) orpreferentially binding to an EphA4 epitope exposed on cancer cells butnot non-cancer cells (e.g., binding poorly to EphA4 that is bound toligand in cell-cell contacts while binding well to EphA4 that is notbound to ligand or in cell-cell contacts) may also be screened.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)2 fragments can also be employed using methods knownin the art such as those disclosed in International Publication No. WO92/22324; Mullinax et al., 1992, BioTechniques 12:864; Sawai et al.,1995, AJRI 34:26; and Better et al., 1988, Science 240:1041 (saidreferences incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing a VHconstant region, e.g., the human gamma 4 constant region, and the PCRamplified VL domains can be cloned into vectors expressing a VL constantregion, e.g., human kappa or lambda constant regions. Preferably, thevectors for expressing the VH or VL domains comprise an EF-1α promoter,a secretion signal, a cloning site for the variable domain, constantdomains, and a selection marker such as neomycin. The VH and VL domainsmay also be cloned into one vector expressing the necessary constantregions. The heavy chain conversion vectors and light chain conversionvectors are then co-transfected into cell lines to generate stable ortransient cell lines that express full-length antibodies, e.g., IgG,using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human or chimericantibodies. Completely human antibodies are particularly desirable fortherapeutic treatment of human subjects. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then be bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, N.J.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715,4,816,567, and 4,816,397, which are incorporated herein by reference intheir entirety. Chimeric antibodies comprising one or more CDRs from anon-human species and framework regions from a human immunoglobulinmolecule can be produced using a variety of techniques known in the artincluding, for example, CDR-grafting (EP 239,400; InternationalPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al.,1994, Protein Engineering 7:805; and Roguska et al., 1994, PNAS 91:969),and chain shuffling (U.S. Pat. No. 5,565,332).

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323,which are incorporated herein by reference in their entireties).

A humanized antibody is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. Ordinarily, the antibody willcontain both the light chain as well as at least the variable domain ofa heavy chain. The antibody also may include the CH1, hinge, CH2, CH3,and CH4 regions of the heavy chain. The humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. Usuallythe constant domain is a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theclass is typically IgG₁. Where such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art. The framework and CDR regions of ahumanized antibody need not correspond precisely to the parentalsequences, e.g., the donor CDR or the consensus framework may bemutagenized by substitution, insertion or deletion of at least oneresidue so that the CDR or framework residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parentalframework region (FR) and CDR sequences, more often 90%, and mostpreferably greater than 95%. Humanized antibodies can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska etal., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332),and techniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,5,585,089, International Publication No. WO 9317105, Tan et al., 2002,J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60,Morea et al., 2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem.272:10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto etal., 1995, Cancer Res. 55 (23 Supp):5973s-5977s, Couto et al., 1995,Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al.,1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525,Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op.Struct. Biol. 2:593-596. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature 332:323, which are incorporated herein byreference in their entireties.)

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438). Theinvention provides methods employing the use of polynucleotidescomprising a nucleotide sequence encoding an antibody of the inventionor a fragment thereof.

5.1.3 Polynucleotides Encoding an Antibody

The methods of the invention also encompass polynucleotides thathybridize under high stringency, intermediate or lower stringencyhybridization conditions, e.g., as defined supra, to polynucleotidesthat encode an antibody of the invention.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Since theamino acid sequences of the antibodies are known, nucleotide sequencesencoding these antibodies can be determined using methods well known inthe art, i.e., nucleotide codons known to encode particular amino acidsare assembled in such a way to generate a nucleic acid that encodes theantibody or fragment thereof of the invention. Such a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY, which areboth incorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence, for example to createamino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the CDRs is inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., 1998, J. Mol. Biol. 278: 457-479 for a listing of human frameworkregions). Preferably, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specificallybinds to EphA4. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

5.1.4 Recombinant Expression of an Antibody

Recombinant expression of an antibody of the invention, derivative,analog or fragment thereof, (e.g., a heavy or light chain of an antibodyof the invention or a portion thereof or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably, but not necessarily, containing the heavyor light chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, a heavy orlight chain of an antibody, a heavy or light chain variable domain of anantibody or a portion thereof, or a heavy or light chain CDR, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,International Publication Nos. WO 86/05807 and WO 89/01036; and U.S.Pat. No. 5,122,464) and the variable domain of the antibody may becloned into such a vector for expression of the entire heavy, the entirelight chain, or both the entire heavy and light chains.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention or fragments thereof, or a heavy or light chain thereof,or portion thereof, or a single chain antibody of the invention,operably linked to a heterologous promoter. In preferred embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains may be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule of the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, NS0, and 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).Preferably, bacterial cells such as Escherichia coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,BioTechnology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding antibodies or fragments thereof whichimmunospecifically bind to EphA4 and agonize EphA4, inhibit a cancercell phenotype, preferentially bind epitopes on EphA4 that areselectively exposed or increased on cancer cells but not non-cancercells and/or have a K_(off) less than 3×10⁻³ s⁻¹ is regulated by aconstitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO12:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, 1984, PNAS 8 1:355-359). Specific initiation signals may also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, NS1 and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, PNAS 77:357; O'Hare et al., 1981, PNAS 78:1527); gpt,which confers resistance to mycophenolic acid (Mulligan & Berg, 1981,PNAS 78:2072); neo, which confers resistance to the aminoglycoside G-418(Wu and Wu, 1991, Biotherapy 3:87; Tolstoshev, 1993, Ann. Rev.Pharmacol. Toxicol. 32:573; Mulligan, 1993, Science 260:926; and Morganand Anderson, 1993, Ann. Rev. Biochem. 62: 191; May, 1993, TIB TECH11:155-); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147). Methods commonly known in the art ofrecombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley& Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1,which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler,1980, PNAS 77:2197). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

5.2 EphA4 Ligand Fusion Proteins

The present invention encompasses the use of fusion proteins comprisingan EphA4 ligand, for example, example Ephrins A1, A2, A3, A4, A5, B2,and B3; B61, AL1/RAGS, LERK4, Htk-L, and Elk-L3, or a ligand fragmentthereof (preferably a fragment that binds to and elicits signaling ofEphA4, recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous agent,particularly the Fc region of an antibody, to generate a fusion protein(see U.S. Pat. No. 5,116,964, by Capan and Lasky). The heterologousagent may be a polypeptide (or portion thereof, preferably a polypeptideof at least 10, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90 or at least 100 aminoacids), nucleic acid, small molecule (less than 1000 daltons), or otherorganic compound. The fusion does not necessarily need to be direct, butmay occur through linker sequences. Such fusion proteins may be used invivo to detect, treat, manage, or monitor the progression of a disorderusing methods known in the art. In a preferred embodiment, EphA4ligand-fusion proteins are used as EphA4 agonists.

5.3 Prophylactic/Therapeutic Methods

The present invention encompasses methods for treating, preventing, ormanaging a disease or disorder associated with overexpression of EphA4and/or cell hyperproliferative disorders, preferably cancer, in asubject comprising administering one or more EphA4 agonistic antibodiesor EphA4 cancer cell phenotype inhibiting antibodies or exposed EphA4epitope antibodies or EphA4 antibodies that bind EphA4 with a K_(off)less than 3×10⁻¹ s⁻¹, preferably one or more monoclonal (or antibodiesfrom some other source of a single antibody species) EphA4 agonisticantibodies or EphA4 cancer cell phenotype inhibiting antibodies orexposed EphA4 epitope antibodies or EphA4 antibodies that bind EphA4with a K_(off) less than 3×10⁻¹ s⁻¹. In a specific embodiment, thedisorder to be treated, prevented, or managed is malignant cancer. Inanother specific embodiment, the disorder to be treated, prevented, ormanaged is pre-malignant cancer.

In one embodiment, the antibodies of the invention can be administeredin combination with one or more other therapeutic agents useful in thetreatment, prevention or management of diseases or disorders associatedwith EphA4 overexpression, hyperproliferative disorders, and/or cancer.In certain embodiments, one or more EphA4 antibodies of the inventionare administered to a mammal, preferably a human, concurrently with oneor more other therapeutic agents useful for the treatment of cancer. Theterm “concurrently” is not limited to the administration of prophylacticor therapeutic agents at exactly the same time, but rather it is meantthat the EphA4 antibodies of the invention and the other agent areadministered to a subject in a sequence and within a time interval suchthat the antibodies of the invention can act together with the otheragent to provide an increased benefit than if they were administeredotherwise. For example, each prophylactic or therapeutic agent may beadministered at the same time or sequentially in any order at differentpoints in time; however, if not administered at the same time, theyshould be administered sufficiently close in time so as to provide thedesired therapeutic or prophylactic effect. Each therapeutic agent canbe administered separately, in any appropriate form and by any suitableroute. In other embodiments, the EphA4 antibodies of the invention areadministered before, concurrently or after surgery. Preferably thesurgery completely removes localized tumors or reduces the size of largetumors. Surgery can also be done as a preventive measure or to relievepain.

In another specific embodiment, the therapeutic and prophylactic methodsof the invention comprise administration of an inhibitor of EphA4expression, such as but not limited to, antisense nucleic acids specificfor EphA4, double stranded EphA4 RNA that mediates RNAi, anti-EphA4ribozymes, etc. (see Section 5.5 infra) or an agonist of EphA4 activityother than an EphA4 antibody, such as small molecule inhibitors oragonists of EphA4 activity.

In a further embodiment, one or more EphA4 agonistic agents of theinvention are administered in combination with one or more EphA2agonistic agents (see U.S. application Ser. No. 10/436,783 by Kinch etal., filed May 12, 2004).

In various embodiments, the prophylactic or therapeutic agents areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, two or more components are administered withinthe same patient visit.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

5.3.1 Patient Population

The invention provides methods for treating, preventing, and managing adisease or disorder associated with EphA4 overexpression and/orhyperproliferative cell disease, particularly cancer, by administratingto a subject in need thereof a therapeutically or prophylacticallyeffective amount of one or more EphA4 antibodies of the invention. Inanother embodiment, the EphA4 antibodies of the invention can beadministered in combination with one or more other therapeutic agents.The subject is preferably a mammal such as non-primate (e.g., cows,pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey, suchas a cynomolgous monkey and a human). In a preferred embodiment, thesubject is a human.

Specific examples of cancers that can be treated by the methodsencompassed by the invention include, but are not limited to, cancersthat over express EphA4. In a further embodiment, the cancer is of anepithelial origin. Examples of such cancers are cancer of the lung,colon, prostate, breast, and skin. Other cancers include cancer of thebladder and pancreas and renal cell carcinoma and melanoma. Additionalcancers are listed by example and not by limitation in the followingsection 5.2.1.1. In particular embodiments, methods of the invention canbe used to treat and/or prevent metastasis from primary tumors.

The methods and compositions of the invention comprise theadministration of one or more EphA4 antibodies of the invention tosubjects/patients suffering from or expected to suffer from cancer,e.g., have a genetic predisposition for a particular type of cancer,have been exposed to a carcinogen, or are in remission from a particularcancer. As used herein, “cancer” refers to primary or metastaticcancers. Such patients may or may not have been previously treated forcancer. The methods and compositions of the invention may be used as afirst line or second line cancer treatment. Included in the invention isalso the treatment of patients undergoing other cancer therapies and themethods and compositions of the invention can be used before any adverseeffects or intolerance of these other cancer therapies occurs. Theinvention also encompasses methods for administering one or more EphA4antibodies of the invention to treat or ameliorate symptoms inrefractory patients. In a certain embodiment, that a cancer isrefractory to a therapy means that at least some significant portion ofthe cancer cells are not killed or their cell division arrested. Thedetermination of whether the cancer cells are refractory can be madeeither in vivo or in vitro by any method known in the art for assayingthe effectiveness of treatment on cancer cells, using the art-acceptedmeanings of “refractory” in such a context. In various embodiments, acancer is refractory where the number of cancer cells has not beensignificantly reduced, or has increased. The invention also encompassesmethods for administering one or more EphA4 agonistic antibodies toprevent the onset or recurrence of cancer in patients predisposed tohaving cancer.

In particular embodiments, the EphA4 antibodies of the invention, orother therapeutics that reduce EphA4 expression, are administered toreverse resistance or reduced sensitivity of cancer cells to certainhormonal, radiation and chemotherapeutic agents thereby resensitizingthe cancer cells to one or more of these agents, which can then beadministered (or continue to be administered) to treat or manage cancer,including to prevent metastasis. In a specific embodiment, EphA4antibodies of the invention are administered to patients with increasedlevels of the cytokine IL-6, which has been associated with thedevelopment of cancer cell resistance to different treatment regimens,such as chemotherapy and hormonal therapy. In another specificembodiment, EphA4 antibodies of the invention are administered topatients suffering from breast cancer that have a decreasedresponsiveness or are refractory to tamoxifen treatment. In anotherspecific embodiment, EphA4 antibodies of the invention are administeredto patients with increased levels of the cytokine IL-6, which has beenassociated with the development of cancer cell resistance to differenttreatment regimens, such as chemotherapy and hormonal therapy.

In alternate embodiments, the invention provides methods for treatingpatients' cancer by administering one or more EphA4 antibodies of theinvention in combination with any other treatment or to patients whohave proven refractory to other treatments but are no longer on thesetreatments. In certain embodiments, the patients being treated by themethods of the invention are patients already being treated withchemotherapy, radiation therapy, hormonal therapy, or biologicaltherapy/immunotherapy. Among these patients are refractory patients andthose with cancer despite treatment with existing cancer therapies. Inother embodiments, the patients have been treated and have no diseaseactivity and one or more agonistic antibodies of the invention areadministered to prevent the recurrence of cancer.

In preferred embodiments, the existing treatment is chemotherapy. Inparticular embodiments, the existing treatment includes administrationof chemotherapies including, but not limited to, methotrexate, taxol,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,dacarbazine, procarbizine, etoposides, campathecins, bleomycin,doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,paclitaxel, docetaxel, etc. Among these patients are patients treatedwith radiation therapy, hormonal therapy and/or biologicaltherapy/immunotherapy. Also among these patients are those who haveundergone surgery for the treatment of cancer.

Alternatively, the invention also encompasses methods for treatingpatients undergoing or having undergone radiation therapy. Among theseare patients being treated or previously treated with chemotherapy,hormonal therapy and/or biological therapy/immunotherapy. Also amongthese patients are those who have undergone surgery for the treatment ofcancer.

In other embodiments, the invention encompasses methods for treatingpatients undergoing or having undergone hormonal therapy and/orbiological therapy/immunotherapy. Among these are patients being treatedor having been treated with chemotherapy and/or radiation therapy. Alsoamong these patients are those who have undergone surgery for thetreatment of cancer.

Additionally, the invention also provides methods of treatment of canceras an alternative to chemotherapy, radiation therapy, hormonal therapy,and/or biological therapy/immunotherapy where the therapy has proven ormay prove too toxic, i.e., results in unacceptable or unbearable sideeffects, for the subject being treated. The subject being treated withthe methods of the invention may, optionally, be treated with othercancer treatments such as surgery, chemotherapy, radiation therapy,hormonal therapy or biological therapy, depending on which treatment wasfound to be unacceptable or unbearable.

In other embodiments, the invention provides administration of one ormore agonistic monoclonal antibodies of the invention without any othercancer therapies for the treatment of cancer, but who have provedrefractory to such treatments. In specific embodiments, patientsrefractory to other cancer therapies are administered one or moreagonistic monoclonal antibodies in the absence of cancer therapies.

In other embodiments, patients with a pre-malignant cancer associatedwith cells that overexpress EphA4 can be administered antibodies of theinvention to treat the disorder and decrease the likelihood that it willprogress to malignant cancer. In a specific embodiments, thepre-malignant cancer is high-grade prostatic intraepithelial neoplasia(PIN), ductal carcinoma of the breast in, or compound nevi.

In yet other embodiments, the invention provides methods of treating,preventing and managing non-cancer hyperproliferative cell disorders,particularly those associated with overexpression of EphA4, includingbut not limited to, asthma, chromic obstructive pulmonary disorder(COPD), restenosis (smooth muscle and/or endothelial), psoriasis, etc.These methods include methods analogous to those described above fortreating, preventing and managing cancer, for example, by administeringthe EphA4 antibodies of the invention, as well as agents that inhibitEphA4 expression, combination therapy, administration to patientsrefractory to particular treatments, etc.

5.3.1.1. Cancers

Cancers and related disorders that can be treated, prevented, or managedby methods and compositions of the present invention include but are notlimited to cancers of an epithelial cell origin. In a preferredembodiment, the cancer is a cancer of the pancreas. Examples of suchcancers include the following: leukemias, such as but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias,such as, myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia leukemias and myelodysplastic syndrome; chronicleukemias, such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors such as but not limited to, glioma, astrocytoma, brain stemglioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer such as but not limited topheochromocytom and adrenocortical carcinoma; thyroid cancer such as butnot limited to papillary or follicular thyroid cancer, medullary thyroidcancer and anaplastic thyroid cancer; pancreatic cancer such as but notlimited to, insulinoma, gastrinoma, glucagonoma, vipoma,somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cycticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, adenocarcinoma,leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers suchas but not limited to squamous cell carcinoma; basal cancers; salivarygland cancers such as but not limited to adenocarcinoma, mucoepidermoidcarcinoma, and adenoidcystic carcinoma; pharynx cancers such as but notlimited to squamous cell cancer, and verrucous; skin cancers such as butnot limited to, basal cell carcinoma, squamous cell carcinoma andmelanoma, superficial spreading melanoma, nodular melanoma, lentigomalignant melanoma, acral lentiginous melanoma; kidney cancers such asbut not limited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America)

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented in the skin, lung, colon, breast,prostate, bladder, kidney, pancreas, ovary, or uterus. In other specificembodiments, sarcoma, melanoma, or leukemia is treated or prevented.

In some embodiments, the cancer is malignant and overexpresses EphA4. Inother embodiments, the cancer is pre-malignant and overexpresses EphA4.In preferred embodiments, the methods and compositions of the inventionare used for the treatment and/or prevention of breast, colon, ovarian,lung, and prostate cancers and melanoma and are provided below byexample rather than by limitation.

5.3.1.2. Treatment of Breast Cancer

In specific embodiments, patients with breast cancer are administered aneffective amount of one or more monoclonal antibodies of the invention.In another embodiment, the antibodies of the invention can beadministered in combination with an effective amount of one or moreother agents useful for breast cancer therapy including but not limitedto: doxorubicin, epirubicin, the combination of doxorubicin andcyclophosphamide (AC), the combination of cyclophosphamide, doxorubicinand 5-fluorouracil (CAF), the combination of cyclophosphamide,epirubicin and 5-fluorouracil (CEF), herceptin, tamoxifen, thecombination of tamoxifen and cytotoxic chemotherapy, taxanes (such asdocetaxel and paclitaxel). In a further embodiment, antibodies of theinvention can be administered with taxanes plus standard doxorubicin andcyclophosphamide for adjuvant treatment of node-positive, localizedbreast cancer.

In a specific embodiment, patients with pre-malignant ductal carcinomaof the breast are administered an EphA4 antibody of the invention totreat the disorder and decrease the likelihood that it will progress tomalignant breast cancer.

5.3.1.3. Treatment of Colon Cancer

In specific embodiments, patients with colon cancer are administered aneffective amount of one or more monoclonal antibodies of the invention.In another embodiment, the antibodies of the invention can beadministered in combination with an effective amount of one or moreother agents useful for colon cancer therapy including but not limitedto: the combination of 5-FU and leucovorin, the combination of 5-FU andlevamisole, irinotecan (CPT-11) or the combination of irinotecan, 5-FUand leucovorin (IFL).

5.3.1.4. Treatment of Prostate Cancer

In specific embodiments, patients with prostate cancer are administeredan effective amount of one or more monoclonal antibodies of theinvention. In another embodiment, the antibodies of the invention can beadministered in combination with an effective amount of one or moreother agents useful for prostate cancer therapy including but notlimited to: external-beam radiation therapy, interstitial implantationof radioisotopes (i.e., I¹²⁵, palladium, iridium), leuprolide or otherLHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide,bicalutamide), steroidal antiandrogens (cyproterone acetate), thecombination of leuprolide and flutamide, estrogens such as DES,chlorotrianisene, ethinyl estradiol, conjugated estrogens U.S.P.,DES-diphosphate, radioisotopes, such as strontium-89, the combination ofexternal-beam radiation therapy and strontium-89, second-line hormonaltherapies such as aminoglutethimide, hydrocortisone, flutamidewithdrawal, progesterone, and ketoconazole, low-dose prednisone, orother chemotherapy regimens reported to produce subjective improvementin symptoms and reduction in PSA level including docetaxel, paclitaxel,estramustine/docetaxel, estramustine/etoposide,estramustine/vinblastine, and estramustine/paclitaxel.

In a specific embodiment, patients with pre-malignant high-gradeprostatic intraepithelial neoplasia (PIN) are administered an EphA4antibody of the invention to treat the disorder and decrease thelikelihood that it will progress to malignant prostate cancer.

5.3.1.5. Treatment of Melanoma

In specific embodiments, patients with melanoma are administered aneffective amount of one or more monoclonal antibodies of the invention.In another embodiment, the antibodies of the invention can beadministered in combination with an effective amount of one or moreother agents useful for melanoma cancer therapy including but notlimited to: dacarbazine (DTIC), nitrosoureas such as carmustine (BCNU)and lomustine (CCNU), agents with modest single agent activity includingvinca alkaloids, platinum compounds, and taxanes, the Dartmouth regimen(cisplatin, BCNU, and DTIC), interferon alpha (IFN-A), and interleukin-2(IL-2). In a specific embodiment, an effective amount of one or moreagonistic monoclonal antibodies of the invention can be administered incombination with isolated hyperthermic limb perfusion (ILP) withmelphalan (L-PAM), with or without tumor necrosis factor-alpha(TNF-alpha) to patients with multiple brain metastases, bone metastases,and spinal cord compression to achieve symptom relief and some shrinkageof the tumor with radiation therapy.

In a specific embodiment, patients with pre-malignant compound nevi areadministered an EphA4 antibody of the invention to treat the disorderand decrease the likelihood that it will progress to malignant melanoma.

5.3.1.6. Treatment of Ovarian Cancer

In specific embodiments, patients with ovarian cancer are administeredan effective amount of one or more monoclonal antibodies of theinvention. In another embodiment, the antibodies of the invention can beadministered in combination with an effective amount of one or moreother agents useful for ovarian cancer therapy including but not limitedto: intraperitoneal radiation therapy, such as P³² therapy, totalabdominal and pelvic radiation therapy, cisplatin, the combination ofpaclitaxel (Taxol) or docetaxel (Taxotere) and cisplatin or carboplatin,the combination of cyclophosphamide and cisplatin, the combination ofcyclophosphamide and carboplatin, the combination of 5-FU andleucovorin, etoposide, liposomal doxorubicin, gemcitabine or topotecan.It is contemplated that an effective amount of one or more agonisticmonoclonal antibodies of the invention is administered in combinationwith the administration Taxol for patients with platinum-refractorydisease. Included is the treatment of patients with refractory ovariancancer including administration of: ifosfamide in patients with diseasethat is platinum-refractory, hexamethylmelamine (HMM) as salvagechemotherapy after failure of cisplatin-based combination regimens, andtamoxifen in patients with detectable levels of cytoplasmic estrogenreceptor on their tumors.

5.3.1.7. Treatment of Lung Cancers

In specific embodiments, patients with small lung cell cancer areadministered an effective amount of one or more monoclonal antibodies ofthe invention. In another embodiment, the antibodies of the inventioncan be administered in combination with an effective amount of one ormore other agents useful for lung cancer therapy including but notlimited to: thoracic radiation therapy, cisplatin, vincristine,doxorubicin, and etoposide, alone or in combination, the combination ofcyclophosphamide, doxorubicin, vincristine/etoposide, and cisplatin(CAV/EP), local palliation with endobronchial laser therapy,endobronchial stents, and/or brachytherapy.

In other specific embodiments, patients with non-small lung cell cancerare administered an effective amount of one or more monoclonalantibodies of the invention in combination with an effective amount ofone or more other agents useful for lung cancer therapy including butnot limited to: palliative radiation therapy, the combination ofcisplatin, vinblastine and mitomycin, the combination of cisplatin andvinorelbine, paclitaxel, docetaxel or gemcitabine, the combination ofcarboplatin and paclitaxel, interstitial radiation therapy forendobronchial lesions or stereotactic radiosurgery.

5.3.2 Other Prophylactic/Therapeutic Agents

In some embodiments, therapy by administration of one or more monoclonalantibodies is combined with the administration of one or more therapiessuch as, but not limited to, chemotherapies, radiation therapies,hormonal therapies, and/or biological therapies/immunotherapies.Prophylactic/therapeutic agents include, but are not limited to,proteinaceous molecules, including, but not limited to, peptides,polypeptides, proteins, including post-translationally modifiedproteins, antibodies etc.; or small molecules (less than 1000 daltons),inorganic or organic compounds; or nucleic acid molecules including, butnot limited to, double-stranded or single-stranded DNA, ordouble-stranded or single-stranded RNA, as well as triple helix nucleicacid molecules. Prophylavtic/therapeutic agents can be derived from anyknown organism (including, but not limited to, animals, plants,bacteria, fungi, and protista, or viruses) or from a library ofsynthetic molecules.

In a specific embodiment, the methods of the invention encompassadministration of a therapeutic antibody of the invention in combinationwith the administration of one or more prophylactic/therapeutic agentsthat are angiogenesis inhibitors such as, but not limited to:Angiostatin (plasminogen fragment); antiangiogenic antithrombin III;Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab; BMS-275291;cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment;CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIIIfragment); fibronectin fragment; Gro-beta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin 16 kD fragment;Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids;Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-β);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD6474;farnesyl transferase inhibitors (FTI); and bisphosphonates.

Additional examples of anti-cancer agents that can be used in thevarious embodiments of the invention, including pharmaceuticalcompositions and dosage forms and kits of the invention, include, butare not limited to: acivicin, aclarubicin, acodazole hydrochloride,acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantroneacetate, aminoglutethimide, amsacrine, anastrozole, anthramycin,asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat,benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate,bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan,cactinomycin, calusterone, caracemide, carbetimer, carboplatin,carmustine, carubicin hydrochloride, carzelesin, cedefingol,chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicinhydrochloride, decarbazine, decitabine, dexormaplatin, dezaguanine,dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicinhydrochloride, droloxifene, droloxifene citrate, dromostanolonepropionate, duazomycin, edatrexate, eflornithine hydrochloride,elsamitrucin, enloplatin, enpromate, epipropidine, epirubicinhydrochloride, erbulozole, esorubicin hydrochloride, estramustine,estramustine phosphate sodium, etanidazole, etoposide, etoposidephosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide,floxuridine, fludarabine phosphate, fluorouracil, flurocitabine,fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride,hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine,interleukin 2 (including recombinant interleukin 2, or rIL2), interferonalpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3,interferon beta-I a, interferon gamma-I b, iproplatin, irinotecanhydrochloride, lanreotide acetate, letrozole, leuprolide acetate,liarozole hydrochloride, lometrexol sodium, lomustine, losoxantronehydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride,megestrol acetate, melengestrol acetate, melphalan, menogaril,mercaptopurine, methotrexate, methotrexate sodium, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolicacid, nitrosoureas, nocodazole, nogalamycin, ormaplatin, oxisuran,paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin sulfate,perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, puromycin, puromycin hydrochloride,pyrazofurin, riboprine, rogletimide, safingol, safingol hydrochloride,semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermaniumhydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin,sulofenur, talisomycin, tecogalan sodium, tegafur, teloxantronehydrochloride, temoporfin, teniposide, teroxirone, testolactone,thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifenecitrate, trestolone acetate, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracilmustard, uredepa, vapreotide, verteporfin, vinblastine sulfate,vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate,vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate,vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin,zinostatin, zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3,5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol,adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine,amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine,anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,antagonist D, antagonist G, antarelix, anti-dorsalizing morphogeneticprotein-1, antiandrogens, antiestrogens, antineoplaston, aphidicolinglycinate, apoptosis gene modulators, apoptosis regulators, apurinicacid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane,atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron,azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat,BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactamderivatives, beta-alethine, betaclamycin B, betulinic acid, bFGFinhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,bistratene A, bizelesin, breflate, bropirimine, budotitane, buthioninesulfoximine, calcipotriol, calphostin C, camptothecin derivatives,canarypox IL-2, capecitabine, carboxamide-amino-triazole,carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor,carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropinB, cetrorelix, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin,cladribine, clomifene analogues, clotrimazole, collismycin A,collismycin B, combretastatin A4, combretastatin analogue, conagenin,crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives,curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide,dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin,diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine,edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin,epristeride, estramustine analogue, estrogen agonists, estrogenantagonists, etanidazole, etoposide phosphate, exemestane, fadrozole,fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,flezelastine, fluasterone, fludarabine, fluorodaunorunicinhydrochloride, forfenimex, formestane, fostriecin, fotemustine,gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix,gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam,heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid,idarubicin, idoxifene, idramantone, ilmofosine, ilomastat,imidazoacridones, imiquimod, immunostimulant peptides, insulin-likegrowth factor-1 receptor inhibitor, interferon agonists, interferons,interleukins, iobenguane, iododoxorubicin, ipomeanol, iroplact,irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,leukemia inhibiting factor, leukocyte alpha interferon,leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole,linear polyamine analogue, lipophilic disaccharide peptide, lipophilicplatinum compounds, lissoclinamide 7, lobaplatin, lombricine,lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine,lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides,maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysininhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone,meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone,miltefosine, mirimostim, mismatched double stranded RNA, mitoguazone,mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonalantibody, human chorionic gonadotrophin, monophosphoryl lipidA+myobacterium cell wall sk, mopidamol, multiple drug resistance geneinhibitor, multiple tumor suppressor 1-based therapy, mustard anticanceragent, mycaperoxide B, mycobacterial cell wall extract, myriaporone,N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip,naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin,nemorubicin, neridronic acid, neutral endopeptidase, nilutamide,nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn,O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone,ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogues,paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid,panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase,peldesine, pentosan polysulfate sodium, pentostatin, pentrozole,perflubron, perfosfamide, perillyl alcohol, phenazinomycin,phenylacetate, phosphatase inhibitors, picibanil, pilocarpinehydrochloride, pirarubicin, piritrexim, placetin A, placetin B,plasminogen activator inhibitor, platinum complex, platinum compounds,platinum-triamine complex, porfimer sodium, porfiromycin, prednisone,propyl bis-acridone, prostaglandin J2, proteasome inhibitors, proteinA-based immune modulator, protein kinase C inhibitor, protein kinase Cinhibitors, microalgal, protein tyrosine phosphatase inhibitors, purinenucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine,pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists,raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors,ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re186 etidronate, rhizoxin, ribozymes, RII retinamide, rogletimide,rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol,saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics,semustine, senescence derived inhibitor 1, sense oligonucleotides,signal transduction inhibitors, signal transduction modulators, singlechain antigen binding protein, sizofiran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosic acid, spicamycin D, spiromustine,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, stromelysin inhibitors, sulfinosine,superactive vasoactive intestinal peptide antagonist, suradista,suramin, swainsonine, synthetic glycosaminoglycans, tallimustine,tamoxifen methiodide, tauromustine, taxol, tazarotene, tecogalan sodium,tegafur, tellurapyrylium, telomerase inhibitors, temoporfin,temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine,thaliblastine, thalidomide, thiocoraline, thioguanine, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin,tirapazamine, titanocene bichloride, topsentin, toremifene, totipotentstem cell factor, translation inhibitors, tretinoin, triacetyluridine,triciribine, trimetrexate, triptorelin, tropisetron, turosteride,tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex,urogenital sinus-derived growth inhibitory factor, urokinase receptorantagonists, vapreotide, variolin B, vector system, erythrocyte genetherapy, velaresol, veramine, verdins, verteporfin, vinorelbine,vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, andzinostatin stimalamer. Preferred additional anti-cancer drugs are5-fluorouracil and leucovorin.

In more particular embodiments, the present invention also comprises theadministration of one or more monoclonal antibodies of the invention incombination with the administration of one or more therapies such as,but not limited to anti-cancer agents such as those disclosed in Table1, preferably for the treatment of breast, ovary, melanoma, prostate,colon and lung cancers as described above.

TABLE 1 Therapeutic Agent Administration Dose Intervals doxorubicinIntravenous 60-75 mg/m² on Day 1 21 day intervals hydrochloride(Adriamycin RDF ® and Adriamycin PFS ®) epirubicin Intravenous 100-120mg/m² on Day 1 of 3-4 week cycles hydrochloride each cycle or dividedequally (Ellence ™) and given on Days 1-8 of the cycle fluorousacilIntravenous How supplied: 5 ml and 10 ml vials (containing 250 and 500mg flourouracil respectively) docetaxel Intravenous 60-100 mg/m² over 1hour Once every 3 weeks (Taxotere ®) paclitaxel Intravenous 175 mg/m²over 3 hours Every 3 weeks for 4 courses (Taxol ®) (administeredsequentially to doxorubicin-containing combination chemotherapy)tamoxifen citrate Oral 20-40 mg Daily (Nolvadex ®) (tablet) Dosagesgreater than 20 mg should be given in divided doses (morning andevening) leucovorin calcium Intravenous or How supplied: Dosage isunclear from text. for injection intramuscular 350 mg vial PDR 3610injection luprolide acetate Single 1 mg (0.2 ml or 20 unit mark) Once aday (Lupron ®) subcutaneous injection flutamide Oral (capsule) 250 mg 3times a day at 8 hour (Eulexin ®) (capsules contain 125 mg intervals(total daily dosage flutamide each) 750 mg) nilutamide Oral 300 mg or150 mg 300 mg once a day for 30 (Nilandron ®) (tablet) (tablets contain50 or 150 mg days followed by 150 mg nilutamide each) once a daybicalutamide Oral 50 mg Once a day (Casodex ®) (tablet) (tablets contain50 mg bicalutamide each) progesterone Injection USP in sesame oil 50mg/ml ketoconazole Cream 2% cream applied once or (Nizoral ®) twicedaily depending on symptoms prednisone Oral Initial dosage may vary from(tablet) 5 mg to 60 mg per day depending on the specific disease entitybeing treated. estramustine Oral 14 mg/kg of body weight Daily given in3 or 4 divided phosphate sodium (capsule) (i.e. one 140 mg capsule fordoses (Emcyt ®) each 10 kg or 22 lb of body weight) etoposide or VP-16Intravenous 5 ml of 20 mg/ml solution (100 mg) dacarbazine Intravenous2-4.5 mg/kg Once a day for 10 days. (DTIC-Dome ®) May be repeated at 4week intervals polifeprosan 20 with wafer placed in 8 wafers, eachcontaining 7.7 mg carmustine implant resection cavity of carmustine, fora total (BCNU) (nitrosourea) of 61.6 mg, if size and shape (Gliadel ®)of resection cavity allows cisplatin Injection How supplied: solution of1 mg/ml in multi- dose vials of 50 mL and 100 mL mitomycin Injectionsupplied in 5 mg and 20 mg vials (containing 5 mg and 20 mg mitomycin)gemcitabine HCl Intravenous For NSCLC-2 schedules 4 week schedule-(Gemzar ®) have been investigated and Days 1,8 and 15 of each 28- theoptimum schedule has not day cycle. Cisplatin been determinedintravenously at 100 mg/m² 4 week schedule- on day 1 after the infusionof administration intravenously Gemzar. at 1000 mg/m² over 30 3 weekschedule- minutes on 3 week schedule- Days 1 and 8 of each 21 day Gemzaradministered cycle. Cisplatin at dosage of intravenously at 1250 mg/m²100 mg/m² administered over 30 minutes intravenously afteradministration of Gemzar on day 1. carboplatin Intravenous Single agenttherapy: Every 4 weeks (Paraplatin ®) 360 mg/m² I.V. on day 1 (infusionlasting 15 minutes or longer) Other dosage calculations: Combinationtherapy with cyclophosphamide, Dose adjustment recommendations, Formuladosing, etc. ifosamide Intravenous 1.2 g/m² daily 5 consecutive days(Ifex ®) Repeat every 3 weeks or after recovery from hematologictoxicity topotecan Intravenous 1.5 mg/m² by intravenous 5 consecutivedays, starting hydrochloride infusion over 30 minutes on day 1 of 21 daycourse (Hycamtin ®) daily

The invention also encompasses administration of the EphA4 antibodies ofthe invention in combination with radiation therapy comprising the useof x-rays, gamma rays and other sources of radiation to destroy thecancer cells. In preferred embodiments, the radiation treatment isadministered as external beam radiation or teletherapy wherein theradiation is directed from a remote source. In other preferredembodiments, the radiation treatment is administered as internal therapyor brachytherapy wherein a radioactive source is placed inside the bodyclose to cancer cells or a tumor mass.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (56th ed., 2002).

5.4 Identification of Antibodies of the Invention

5.4.1 Agonistic Antibodies

Antibodies of the invention may preferably agonize (i.e., elicit EphA4phosphorylation) as well as immunospecifically bind to the EphA4receptor. When agonized, EphA4 phosphorylates a 75 kDa EphA4-associatedprotein and may itself be phosphorylated and then subsequently degraded.Any method known in the art to assay either the level of EphA4 or 75 kDaEphA4-associated protein phosphorylation, activity, or expression can beused to assay candidate EphA4 antibodies to determine their agonisticactivity (see, e.g., Section 6.2 infra).

5.4.2 Antibodies that Preferentially Bind EphA4 Epitopes Exposed onCancer Cells

Antibodies of the invention may preferably bind to EphA4 epitopesexposed on cancer cells (e.g., cells overexpressing EphA4 and/or cellswith substantial EphA4 that is not bound to ligand) but not non-cancercells or cells where EphA4 is bound to ligand. In this embodiment,antibodies of the invention are antibodies directed to an EphA4 epitopenot exposed on non-cancer cells but exposed on cancer cells.

Any method known in the art to determine candidate EphA4 antibodybinding/localization on a cell can be used to screen candidateantibodies for desirable binding properties. In a one embodiment,immunofluorescence microscopy is used to determine the bindingcharacteristics of an antibody. Standard techniques can be used tocompare the binding of an antibody binding to cells grown in vitro. In aspecific embodiment, antibody binding to cancer cells is compared toantibody binding to non-cancer cells. An exposed EphA4 epitope antibodybinds poorly to non-cancer cells but binds well to cancer cells. Inanother specific embodiment, antibody binding to non-cancer dissociatedcells (e.g., treated with a calcium chelator such as EGTA) is comparedto antibody binding to non-cancer cells that have not been dissociated.An exposed EphA4 epitope antibody binds poorly non-cancer cells thathave not been dissociated but binds well to dissociated non-cancercells.

In another embodiment, flow cytometry is used to determine the bindingcharacteristics of an antibody. In this embodiment, EphA4 may or may notbe crosslinked to its ligand. An exposed EphA4 epitope antibody bindspoorly crosslinked EphA4 but binds well to uncrosslinked EphA4.

In another embodiment, cell-based or immunoassays are used to determinethe binding characteristics of an antibody. In this embodiment,antibodies that can compete with an EphA4 ligand (e.g., Ephrins A1, A2,A3, A4, A5, B2, and B3; B61, AL1/RAGS, LERK4, Htk-L, and Elk-L3) forbinding to EphA4 displace the ligand from EphA4. The EphA4 ligand usedin this assay can be soluble protein (e.g., recombinantly expressed) orexpressed on a cell so that it is anchored to the cell.

5.4.3 Cancer Cell Phenotype Inhibiting Antibodies

Antibodies of the invention may preferably inhibit (and preferablyreduce) cancer cell colony formation in, for example, soft agar, ortubular network formation in a three-dimensional basement membrane orextracellular matrix preparation as well as immunospecifically bind tothe EphA4 receptor. One of skill in the art can assay candidate EphA4antibodies for their ability to inhibit such behavior (see, e.g.,Section 6.3 infra). Metastatic tumor cells suspended in soft agar formcolonies while benign tumors cells do not. Colony formation in soft agarcan be assayed as described in Zelinski et al. (2001, Cancer Res.61:2301-6, incorporated herein by reference in its entirety). Antibodiesto be assayed for agonistic activity can be included in bottom and topagar solutions. Metastatic tumor cells can be suspended in soft agar andallowed to grow. EphA4 cancer cell phenotype inhibiting antibodies willinhibit colony formation.

Another behavior specific to metastatic cells that can be used toidentify cancer cell phenotype inhibiting antibodies is tubular networkformation within a three-dimensional microenvironment, such asMATRIGEL™. Normally, cancer cells quickly assemble into tubular networksthat progressively invade all throughout the MATRIGEL™. In the presenceof an EphA4 cancer cell phenotype inhibiting antibody, cancer cellsassemble into spherical structures that resemble the behavior ofdifferentiated, non-cancerous cells. Accordingly, EphA4 cancer cellphenotype inhibiting antibodies can be identified by their ability toinhibit tubular network formation of cancer cells.

Any other method that detects an increase in contact inhibition of cellproliferation (e.g., reduction of colony formation in a monolayer cellculture) may also be used to identify cancer cell phenotype inhibitingantibodies.

In addition to inhibiting cancer cell colony formation, cancer cellphenotype inhibiting antibodies may also cause a reduction orelimination of colonies when added to already established colonies ofcancer cells by cell killing, e.g., by necrosis or apoptosis. Methodsfor assaying for necrosis and apoptosis are well known in the art.

5.4.4 Antibodies with Low K_(off) Rates

The binding affinity of a monoclonal antibody of the invention to EphA4or a fragment thereof and the off-rate of a monoclonal antibody-EphA4interaction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled EphA4 (e.g., ³H or ¹²⁵I) with the monoclonalantibody of interest in the presence of increasing amounts of unlabeledEphA4, and the detection of the monoclonal antibody bound to the labeledEphA4. The affinity of a monoclonal antibody for an EphA4 and thebinding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second monoclonal antibody can also bedetermined using radioimmunoassays. In this case, EphA4 is incubatedwith a monoclonal antibody conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of a second unlabeledmonoclonal antibody.

In a preferred embodiment, a candidate EphA4 antibody may be assayedusing any surface plasmon resonance based assays known in the art forcharacterizing the kinetic parameters of the EphA4-EphA4 antibodyinteraction. Any SPR instrument commercially available including, butnot limited to, BIACORE Instruments, available from Biacore AB (Uppsala,Sweden); IAsys instruments available form Affinity Sensors (Franklin,Mass.); IBIS system available from Windsor Scientific Limited (Berks,UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab(Hokkaido, Japan), and SPR Detector Spreeta available from TexasInstruments (Dallas, Tex.) can be used in the instant invention. For areview of SPR-based technology see Mullet et al., 2000, Methods 22:77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash etal., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al.,2000, Current Opinion in Biotechnology 11: 54-61; all of which areincorporated herein by reference in their entirety. Additionally, any ofthe SPR instruments and SPR based methods for measuring protein-proteininteractions described in U.S. Pat. Nos. 6,373,577; 6,289,286;5,322,798; 5,341,215; 6,268,125 are contemplated in the methods of theinvention, all of which are incorporated herein by reference in theirentirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution. SPR is based on measuring the change inrefractive index of the solvent near the surface that occurs uponcomplex formation or dissociation. The surface onto which theimmobilization occur is the sensor chip, which is at the heart of theSPR technology; it consists of a glass surface coated with a thin layerof gold and forms the basis for a range of specialized surfaces designedto optimize the binding of a molecule to the surface. A variety ofsensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups and attachmentthrough the histidine tag with NTA chips.

In a more preferred embodiment, BIACORE™ kinetic analysis is used todetermine the binding on and off rates of monoclonal antibodies to EphA4(see, e.g., Section 6.5 infra). BIACORE™ kinetic analysis comprisesanalyzing the binding and dissociation of a monoclonal antibody fromchips with immobilized EphA4 or fragment thereof on their surface.

Once an entire data set is collected, the resulting binding curves areglobally fitted using computer algorithms supplied by the manufacturer,BIAcore, Inc. (Piscataway, N.J.). These algorithms calculate both theK_(on) and K_(off), from which the apparent equilibrium bindingconstant, K_(D) is deduced as the ratio of the two rate constants (i.e.,K_(off)/K_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BIAevaluaion Software Handbook(BIAcore, Inc., Piscataway, N.J.). The analysis of the generated datamay be done using any method known in the art. For a review of thevarious methods of interpretation of the kinetic data generated seeMyszka, 1997, Current Opinion in Biotechnology 8: 50-7; Fisher et al.,1994, Current Opinion in Biotechnology 5: 389-95; O'Shannessy, 1994,Current Opinion in Biotechnology, 5:65-71; Chaiken et al., 1992,Analytical Biochemistry, 201: 197-210; Morton et al., 1995, AnalyticalBiochemistry 227: 176-85; O'Shannessy et al., 1996, AnalyticalBiochemistry 236: 275-83; all of which are incorporated herein byreference in their entirety.

An antibody that immunospecifically binds to EphA4 preferably has aK_(off) rate

of less than 3×10⁻³ s⁻¹, less than 10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹, lessthan 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, lessthan 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

Thus, the invention provides methods of assaying and screening for EphA4antibodies of the invention by incubating antibodies that specificallybind EphA4, particularly that bind the extracellular domain of EphA4,with cells that express EphA4, particularly cancer cells, preferablymetastatic cancer cells, that overexpress EphA4 (relative to non-cancercells of the same cell type) and then assaying for an increase in EphA4phosphorylation and/or EphA4 degradation (for agonistic antibodies), orreduction in colony formation in soft agar or tubular network formationin three-dimensional basement membrane or extracellular matrixpreparation (for cancer cell phenotype inhibiting antibodies), orincreased antibody binding to cancer cells as compared to non-cancercells by e.g., immunofluorescence (for exposed EphA4 epitope antibodies)thereby identifying an EphA4 antibody of the invention.

5.5 Nucleic Acid Molecules

In addition to EphA4 antibodies of the invention, nucleic acid moleculesspecific for EphA4 can also be used to decrease EphA4 expression and,therefore, be used in methods of the invention.

5.5.1 Antisense

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to all or part of a sensenucleic acid encoding EphA4, e.g., complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can be antisense to all or part of a non-coding region ofthe coding strand of a nucleotide sequence encoding a polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleicacid of the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine,inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,β-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, i.e., EphA4).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a selectedpolypeptide of the invention to thereby inhibit expression, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131) or a chimeric RNA-DNAanalogue (Inoue et al., 1987, FEBS Lett. 215:327).

5.5.2 Ribozymes

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes;described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding EphA4 can be designedbased upon the nucleotide sequence of EphA4. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in U.S. Pat. Nos. 4,987,071 and 5,116,742. Alternatively,an mRNA encoding a polypeptide of the invention can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak, 1993, Science 261:1411.

5.4.3 RNA Interference

In certain embodiments, an RNA interference (RNAi) molecule is used todecrease EphA4 expression. RNA interference (RNAi) is defined as theability of double-stranded RNA (dsRNA) to suppress the expression of agene corresponding to its own sequence. RNAi is also calledpost-transcriptional gene silencing or PTGS. Since the only RNAmolecules normally found in the cytoplasm of a cell are molecules ofsingle-stranded mRNA, the cell has enzymes that recognize and cut dsRNAinto fragments containing 21-25 base pairs (approximately two turns of adouble helix and which are referred to as small interfering RNA orsiRNA). The antisense strand of the fragment separates enough from thesense strand so that it hybridizes with the complementary sense sequenceon a molecule of endogenous cellular mRNA. This hybridization triggerscutting of the mRNA in the double-stranded region, thus destroying itsability to be translated into a polypeptide. Introducing dsRNAcorresponding to a particular gene thus knocks out the cell's ownexpression of that gene in particular tissues and/or at a chosen time.

Double-stranded (ds) RNA can be used to interfere with gene expressionin mammals (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2: 70-75;incorporated herein by reference in its entirety). dsRNA is used asinhibitory RNA or RNAi of the function of EphA4 to produce a phenotypethat is the same as that of a null mutant of EphA4 (Wianny &Zernicka-Goetz, 2000, Nature Cell Biology 2: 70-75).

5.6 Characterization and Demonstration of Therapeutic or ProphylacticUtility

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice where a mouse EphA4 is replaced with the human EphA4,nude mice with human xenografts, animal models, such as hamsters,rabbits, etc. known in the art and described in Relevance of TumorModels for Anticancer Drug Development (1999, eds. Fiebig and Burger);Contributions to Oncology (1999, Karger); The Nude Mouse in OncologyResearch (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

5.6.1 Demonstration of Therapeutic or Prophylactic Utility

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. For example, in vitro assays which canbe used to determine whether administration of a specific therapeuticprotocol is indicated, include in vitro cell culture assays in which apatient tissue sample is grown in culture, and exposed to or otherwiseadministered a protocol, and the effect of such protocol upon the tissuesample is observed, e.g., increased phosphorylation or degradation ofEphA4, inhibition of or decrease in growth and/or colony formation insoft agar or tubular network formation in three-dimensional basementmembrane or extracellular matrix preparations. A lower level ofproliferation or survival of the contacted cells indicates that thetherapeutic agent is effective to treat the condition in the patient.Alternatively, instead of culturing cells from a patient, therapeuticagents and methods may be screened using cells of a tumor or malignantcell line. Many assays standard in the art can be used to assess suchsurvival and/or growth; for example, cell proliferation can be assayedby measuring ³H-thymidine incorporation, by direct cell count, bydetecting changes in transcriptional activity of known genes such asproto-oncogenes (e.g., fos, myc) or cell cycle markers; cell viabilitycan be assessed by trypan blue staining, differentiation can be assessedvisually based on changes in morphology, increased phosphorylation ordegradation of EphA4, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer.

5.7 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of one or more EphA4 antibodies of theinvention and a pharmaceutically acceptable carrier or an agent thatreduces EphA4 expression (e.g., antisense oligonucleotides) and apharmaceutically acceptable carrier. In a further embodiment, thecomposition of the invention further comprises an additionaltherapeutic, e.g., anti-cancer, agent.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete) or, more preferably, MF59C.1 adjuvantavailable from Chiron, Emeryville, Calif.), excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Various delivery systems are known and can be used to administer anagonistic monoclonal antibody of the invention or the combination of anagonistic monoclonal antibody of the invention and a prophylactic agentor therapeutic agent useful for preventing or treating cancer, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the antibody or antibody fragment,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of administering a prophylacticor therapeutic agent of the invention include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal, inhaled, and oral routes). In a specific embodiment,prophylactic or therapeutic agents of the invention are administeredintramuscularly, intravenously, or subcutaneously. The prophylactic ortherapeutic agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.

In yet another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the antibodies ofthe invention or fragments thereof (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos.5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; InternationalPublication Nos. WO 99/15154 and WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

5.7.1 Gene Therapy

In a specific embodiment, nucleic acids that reduce EphA4 expression(e.g., EphA4 antisense nucleic acids or EphA4 dsRNA) are administered totreat, prevent or manage cancer by way of gene therapy. Gene therapyrefers to therapy performed by the administration to a subject of anexpressed or expressible nucleic acid. In this embodiment of theinvention, the antisense nucleic acids are produce and mediate aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488; Wu and Wu, 1991, Biotherapy 3:87;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573; Mulligan, 1993,Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191; May, 1993, TIBTECH 11:155. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); and Kriegler, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises EphA4nucleic acids that reduce EphA4 expression, said nucleic acids beingpart of an expression vector that expresses the nucleic acid in asuitable host. In particular, such nucleic acids have promoters,preferably heterologous promoters, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the nucleic acidthat reduces EphA4 expression and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe nucleic acids that reduce EphA4 expression (Koller and Smithies,1989, PNAS 86:8932; Zijlstra et al., 1989, Nature 342:435).

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy. In a specific embodiment, the nucleic acidsequences are directly administered in vivo. This can be accomplished byany of numerous methods known in the art, e.g., by constructing them aspart of an appropriate nucleic acid expression vector and administeringit so that they become intracellular, e.g., by infection using defectiveor attenuated retrovirals or other viral vectors (see U.S. Pat. No.4,980,286), or by direct injection of naked DNA, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents,encapsulation in liposomes, microparticles, or microcapsules, or byadministering them in linkage to a peptide which is known to enter thenucleus, by administering it in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429) (which can be used to target cell types specificallyexpressing the receptors), etc. In another embodiment, nucleicacid-ligand complexes can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., International Publication Nos.WO 92/06180; WO 92/22635; WO92/203 16; WO93/14188, WO 93/20221).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, PNAS 86:8932; and Zijlstra etal., 1989, Nature 342:435).

In a specific embodiment, viral vectors that contain the nucleic acidsequences that reduce EphA4 expression are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581). These retroviral vectors contain the components necessary forthe correct packaging of the viral genome and integration into the hostcell DNA. The nucleic acid sequences to be used in gene therapy arecloned into one or more vectors, which facilitates delivery of thenucleic acid into a subject. More detail about retroviral vectors can befound in Boesen et al., 1994, Biotherapy 6:291-302, which describes theuse of a retroviral vector to deliver the mdr 1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest.93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Adenoviruses have theadvantage of being capable of infecting non-dividing cells. Kozarsky andWilson, 1993, Current Opinion in Genetics Development 3:499 present areview of adenovirus-based gene therapy. Bout et al., 1994, Human GeneTherapy 5:3-10 demonstrated the use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys. Other instances ofthe use of adenoviruses in gene therapy can be found in Rosenfeld etal., 1991, Science 252:431; Rosenfeld et al., 1992, Cell 68:143;Mastrangeli et al., 1993, J. Clin. Invest. 91:225; InternationalPublication No. WO94/12649; and Wang et al., 1995, Gene Therapy 2:775.In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; andU.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599; Cohen et al., 1993, Meth. Enzymol. 217:618) and may beused in accordance with the present invention, provided that thenecessary developmental and physiological functions of the recipientcells are not disrupted. The technique should provide for the stabletransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and preferably heritable and expressible by itscell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. The amount of cells envisioned for use dependson the desired effect, patient state, etc., and can be determined by oneskilled in the art.

5.7.2 Formulations

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the EphA4 agonistic antibodies of the invention or otheranti-EphA4 agents (e.g., antisense and other nucleic acids) and theirphysiologically acceptable salts and solvates may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, parenteral or mucosal (such as buccal, vaginal,rectal, sublingual) administration. In a preferred embodiment, local orsystemic parenteral administration is used.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the prophylactic or therapeutic agentsfor use according to the present invention are conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The prophylactic or therapeutic agents may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The prophylactic or therapeutic agents may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the prophylacticor therapeutic agents may also be formulated as a depot preparation.Such long acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the prophylactic or therapeutic agents maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The invention also provides that a prophylactic or therapeutic agent ispackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity. In one embodiment, the prophylactic ortherapeutic agent is supplied as a dry sterilized lyophilized powder orwater free concentrate in a hermetically sealed container and can bereconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject.

In a preferred embodiment of the invention, the formulation andadministration of various chemotherapeutic, biological/immunotherapeuticand hormonal therapeutic agents are known in the art and often describedin the Physician's Desk Reference, 56^(th) ed. (2002). For instance, incertain specific embodiments of the invention, the therapeutic agents ofthe invention can be formulated and supplied as provided in Table 1.

In other embodiments of the invention, radiation therapy agents such asradioactive isotopes can be given orally as liquids in capsules or as adrink. Radioactive isotopes can also be formulated for intravenousinjections. The skilled oncologist can determine the preferredformulation and route of administration.

In certain embodiments the agonistic monoclonal antibodies of theinvention, are formulated at 1 mg/ml, 5 mg/ml, 10 mg/ml, and 25 mg/mlfor intravenous injections and at 5 mg/ml, 10 mg/ml, and 80 mg/ml forrepeated subcutaneous administration and intramuscular injection.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

5.7.3 Dosages

The amount of the composition of the invention which will be effectivein the treatment, prevention or management of cancer can be determinedby standard research techniques. For example, the dosage of thecomposition which will be effective in the treatment, prevention ormanagement of cancer can be determined by administering the compositionto an animal model such as, e.g., the animal models disclosed herein orknown to those skilled in the art. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges.

Selection of the preferred effective dose can be determined (e.g., viaclinical trials) by a skilled artisan based upon the consideration ofseveral factors which will be known to one of ordinary skill in the art.Such factors include the disease to be treated or prevented, thesymptoms involved, the patient's body mass, the patient's immune statusand other factors known by the skilled artisan to reflect the accuracyof administered pharmaceutical compositions.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the cancer, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human and humanized antibodies have alonger half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible.

For other cancer therapeutic agents administered to a patient, thetypical doses of various cancer therapeutics known in the art areprovided in Table 1. Given the invention, certain preferred embodimentswill encompass the administration of lower dosages in combinationtreatment regimens than dosages recommended for the administration ofsingle agents.

The invention provides for any method of administrating lower doses ofknown prophylactic or therapeutic agents than previously thought to beeffective for the prevention, treatment, management or amelioration ofcancer. Preferably, lower doses of known anti-cancer therapies areadministered in combination with lower doses of agonistic monoclonalantibodies of the invention.

5.8 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with an monoclonal antibody of the invention.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a cancer can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more a monoclonalantibodies of the invention. In another embodiment, a kit furthercomprises one or more other prophylactic or therapeutic agents usefulfor the treatment of cancer, in one or more containers. In certainembodiments, the other prophylactic or therapeutic agent is achemotherapeutic. In other embodiments, the prophylactic or therapeuticagent is a biological or hormonal therapeutic.

6. EXAMPLES

6.1 EphA4 Antibodies Cause Changes in Cell Behavior

Cell Adhesion and Cell Rounding Assays

To examine cell rounding, pancreatic cancer cell line Aspc1 cells wereplated onto six-well dishes previously coated with extracellularmembrane (ECM) proteins, or plated onto ECM protein-coated coverslips in24-well dishes. Cells were allowed to adhere for 48 hours, thenincubated with media containing EphA4 scFv antibody at 37° C. As acontrol, sets of cells plated and allowed to adhere as above wereincubated with EphA4 scFv antibody at 0° C. rather than 37° C. Followingincubation, the EphA4-scFv incubated cells were washed and treated withanti-Flag antibodies to cross-link EphA4-bound scFv. Plates orcoverslips were washed, fixed, stained, and visualized by microscopy.Cells incubated with EphA4 antibody at 37° C. show cell roundingrelative to cells incubated with EphA4 antibody at 0° C., i.e. atemperature at which the cells will not change shape (FIGS. 1A,B). Thisfurther indicates that anti-EphA4 antibodies cause decreased attachmentto the ECM matrix. Decreased ECM-cell attachment is significant sincethese types of adhesions are sites of intracellular signaling thatpromote cell growth, survival, migration and invasion (Burridge andChrzanowska-Wodnicka, Annu. Rev. Cell Dev. Biol., 12: 463, 1996;Parsons, Curr. Opin. Cell Biol., 8:146, 1996).

Cell adhesion and cell rounding assays can also be performed asdescribed in Miao, et al. (Nature Cell Biol. 2:62, 2000), which isincorporated by reference herein in its entirety. To study celladhesion, briefly, cells are plated in triplicate onto 96-well platespreviously coated with various ECM proteins or poly-L-lysine. Cells areplated at a density of 1×10⁵ cells per well in the presence or absenceof EphA4 antibodies (or other EphA4 agonist) and allowed to adhere for30 minutes at 37° C. Non-adherent cells are washed from the wells, andadherent cells are fixed, stained, and quantified by measuringabsorbance on an enzyme-linked immunosorbent assay (ELISA) reader. Cellstreated with EphA4 antibodies show decreases in attachment to ECMprotein-treated wells relative to control cells allowed to adhere in theabsence of EphA4 antibodies.

For cell rounding assays, briefly, cells are plated onto ECMprotein-coated six-well dishes, or ECM protein-coated coverslips in24-well dishes. Cells are allowed to adhere for 48 hours, then treatedwith media with or without EphA4 antibody (or other EphA4 agonist) for10 minutes. Plates or coverslips are washed, fixed, stained, andvisualized by microscopy. Cells treated with EphA4 antibody show cellrounding relative to cells treated with media lacking EphA4 antibody,indicating decreased attachment to the ECM matrix.

6.2 Preparation of Monoclonal Antibodies

Immunization and Fusion

Monoclonal antibodies against the extracellular domain of EphA4 aregenerated using the fusion protein EphA4-Fc. This fusion proteinconsists of the extracellular domain of human EphA4 linked to humanimmunoglobulin to facilitate secretion of the fusion protein.

Mice (either Balb/c mice or SJL mice) are injected with 5 μg of EphA4fusion protein in TiterMax Adjuvant (total volume 100 μl) in the leftmetatarsal region at days 0 and 7. Mice are injected with 10 μg of EphA4fusion protein in PBS (total volume 100 μl) in the left metatarsalregion at days 12 and 14. On day 15, the popliteal and inguinal lymphnode cells from the left leg and groin are removed and somatically fused(using PEG) with P3XBcl-2-13 cells.

Hybridomas producing EphA4 antibody are isolated from fusions of lymphnodes from immunized SJL mice.

Antibody Screening

Supernatants from bulk culture hybridomas can be screened forimmunoreactivity against EphA4 using standard molecular biologicaltechniques (e.g., ELISA immunoassay). Supernatants are further screenedfor the ability to inhibit an EphA4 monoclonal antibody from binding toEphA4. Briefly, the ability of labeled EphA4 antibody to bind EphA4fusion protein is assayed by competitive ELISA in presence of eitherunlabeled EphA4 antibody or other unlabeled EphA4 binding protein. Thesemay decrease the amount of labeled EA4 binding to EphA4-Fc withincreasing concentrations of unlabeled antibody or binding proteinadded.

6.3 Use of EphA4 Antibodies

6.3.1 EphA4 Phosphorylation

EphA4 antibodies promoted tyrosine phosphorylation in ASPC1 cells. Cellswere incubated in the presence of an anti-EphA4 monoclonal antibody (NED154-567) or control for 15 minutes at 37° C. Cell lysates were thenimmunoprecipitated with EphrinA4-Fc (R&D Systems; Minneapolis, Minn.),resolved by SDS-PAGE, and subjected to western blot analysis with acocktail of the anti-phosphotyrosine antibodies 4G10 (UpstateBiotechnology; Lake Placid N.Y.) and PY20 (BD Transduction Laboratories;Franklin Lakes, N.J.). Increased EphA4 phosphorylation was seenfollowing treatment with EphA4 antibodies, indicating that the EphA4antibodies agonize EphA4 and likely promote EphA4 auto-phosphorylation.

EphA4 scFv antibody clone EA44 stongly promoted tyrosine phosphorylationof EphA4 (FIG. 6). Cells that express the anti-EphA4 scFv EA44 have beendeposited with the American Type Culture Collection (P.O. Box 1549,Manassas, Va. 20108) on Jun. 1, 2004 under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedures, and assignedaccession number ______. The VH and VL CDR amino acid sequences of EA44are contained in SEQ IDs NO: 22, 24, 26, 28, 30, and 32 respectively(FIG. 7). Aspc1 pancreatic carcinoma cells were incubated with theindicated anti-EphA4 scFv (5 μg/ml) on ice. After 30 minutes, medium wasremoved, cells were washed with ice-cold PBS, and medium containing 10μg/ml antibody specific for the flag epitope M2 were placed onto cells.At this time, a control sample was treated with 10 μg/ml mAb9, anantibody known to induce EphA4 tyrosine phosphorylation. Cells wereincubated with scFv clones 8, 18, 20, 36, 41, or 44, or mAb9 for 15minutes at 37° C., washed with ice-cold PBS, and then lysed inTris-buffered saline containing 1% Triton X-100. EphA4 wasimmunoprecipitated from the cell lysates using ephrinA4-Fc (4 μg; R&DSystems), resolved by SDS-PAGE, and transferred to nitrocellulose. Thestatus of EphA4 tyrosine phosphorylation was analyzed by western blotanalysis using the anti-pTyr antibodies, 4G10 and PY20. As seen in FIG.6, scFv clone 44 strongly promotes EphA4 phosphorylation.

EphA4 antibodies also promoted tyrosine phosphorylation of anEphA4-associated protein. Cells were incubated with LX13 scFv antibody,washed, and subsequently incubated with anti-Flag antibodies tocrosslink scFv on the surface of cells. EphA4 was thenimmunoprecipitated with EphrinA4-Fc, and western blotting was performedwith anti-phosphotyrosine antibodies. As seen in FIG. 2, crosslinking ofEphA4 (“X-linked EphA4” lane) causes increased tyrosine phosphorylationof an EphA4-associated protein of approximately 75 kDa relative tophosphorylation of this EphA4-associated protein of approximately 75 kDain cells in which EphA4 is not crosslinked (“Control” lane).

6.3.2. EphA4 Degradation

Antibody-mediated degradation of EphA4 can also be assessed. Monolayersof EphA4-positive cells, such as the pancreatic carcinoma cell linesASPC1 or MiaPaca2, are incubated in the presence of EphA4 antibodies (10μg/ml) at 37° C. Cells are then lysed in Tris-buffered saline containing1% Triton X-100 (Sigma, St. Louis, Mo.) at the following timepointsafter addition of antibody: 0, 0.5. 1, 2, 4, 8, and 24 hours. Aftermeasuring protein concentrations (BioRad, Hercules, Calif.), equalquantities of total protein are resolved by SDS-PAGE and transferred tonitrocellulose (Protran™, Schleicher and Schuell, Keene, NH). EphA4levels are visualized via western blot analysis using a commerciallyavailable EphA4/Sek monoclonal antibody (BD Biosciences, San Jose,Calif.). Membranes are then stripped and reprobed with apaxillin-specific antibody (clone 5H11; Upstate Biotechnology, LakePlacid, N.Y.) to assure equal loading of total protein between samples.

6.3.3. Growth in Soft Agar

The ability of the antibodies of the invention to inhibit cancer cellformation in soft agar was assayed (such assays may be carried out,e.g., as described in Zelinski et al., 2001, Cancer Res. 61:2301,incorporated by reference herein in its entirety). Briefly, cells weresuspended in soft agar for 7 days at 37° C. in the presence ofanti-EphA4 LX13 scFv antibody (LX13), or control solution (PBS).Following incubation with LX13 or PBS, cells were washed and incubatedwith either an anti-LX13 secondary monoclonal antibody (secondary mab)or PBS. Colony formation was scored microscopically. Clusters containingat least three cells were scored as a positive. As seen in FIG. 3, cellsincubated with LX13 and secondary mab (“LX13 bival”), which containaggregated EphA4, show significant inhibition of growth in soft agarrelative to cells incubated with PBS followed by secondary mab alone(“Control”) or cells treated with LX13 followed by PBS (“LX13”).

The ability of the antibodies of the invention to eliminate cancer cellcolonies already formed in soft agar is assayed. Assay methods aresimilar to those described above except that antibodies are not added tothe cancer cells until the third day of growth in soft agar.

6.3.4. Estrogen Dependence in Breast Cancer Cells

Estrogen-sensitive breast cancer cells, MCF-7 cells, were transfectedwith and stably overexpress human EphA4 (MCF-7^(EphA4)). Western blotanalyses confirm the ectopic overexpression of EphA4 in transfectedcells relative to matched controls (data not shown). TwoEphA4-overexpressing clones, EphA4-2 and EphA4-3, were selected forfurther study.

EphA4 overexpression increases malignant growth and decreases the effectof estrogen. Colony formation in soft agar of the EphA4-2 and EphA4-3clones and vector alone control were assayed in the presence or absenceof estrogen.

In the presence of estrogen, the EphA4-2 and EphA4-3 cells demonstrate agreater than two-fold increase in colony formation in soft agar relativeto matched controls (FIG. 4, “+Estrogen”). EphA4-2 and EphA4-3 cellsform an average of 4.6 colonies/field (FIG. 4, “+Estrogen”, “EphA4-2”and “EphA4-3” bars), while control cells transfected with empty vectorform fewer than 2 colonies/field (FIG. 4, “+Estrogen”, “Vector” bar).

Parallel studies are performed in the absence of exogenous estrogen(FIG. 4, “−Estrogen” bars). Experimental deprivation of estrogenamplifies differences between the cellular behaviors of control andEphA4-2 and EphA4-3 cells. Although MCF-7 control cells are largelyunable to colonize soft agar (FIG. 4, “Vector” bar in “−Estrogen” field,<1 colony/field), EphA4-2 and EphA4-3 cells form more numerous colonies(3.4 colonies/field; FIG. 4, “−Estrogen” bars). While EphA4-2 andEphA4-3 cells colonize soft agar more efficiently than matched controls,these cells grow well in the absence of exogenous estrogen (FIG. 4,“−Estrogen” bars). Thus, EphA4 overexpression decreases the need forexogenous estrogen.

6.3.5. Tubular Network Formation in MATRIGEL™

Tumor cell behavior within a three-dimensional microenvironment, such asMATRIGEL™, can reliably predict the differentiation state andaggressiveness of breast epithelial cells. Monolayer cultures of benign(MCF-10A) or malignant (MDA-MB-231) breast epithelial cells areincubated on MATRIGEL™ in the presence of EphA4 antibodies (10 μg/ml) orcontrol solution (PBS). The behavior of cells on MATRIGEL™ is analyzedas described in Zelinski et al. (2001, Cancer Res. 61:2301-6). Briefly,tissue culture dishes are coated with MATRIGEL™ (CollaborativeBiomedical Products, Bedford, Mass.) at 37° C. before adding 1×10⁵MDA-MB-231 or MCF-10A cells previously incubated on ice for 1 hour withthe EphA4 antibody or control solution (PBS). Cells are incubated onMATRIGEL™ for 24 hours at 37° C., and cell behavior is assessed using anOlympus IX-70 inverted light microscope. All images are recorded onto 35mm film (T-Max-400. Kodak, Rochester, N.Y.).

6.3.6. Growth in vivo

The ability of the antibodies of the invention to inhibit tumor cancergrowth in vivo is assayed. Briefly, MDA-MB-231 breast cancer cells areimplanted subcutaneously into athymic mice. After the tumors have grownto an average volume of 100 mm³, mice are administered 6 mg/ml EphA4antibody or PBS control intraperitoneally twice a week for 3 weeks.Tumor growth is assessed and expressed as a ratio of the tumor volumedivided by initial tumor volume (100 mm³). Tumor growth is allowed toproceed until tumor volume reaches 1000 mm³. Survival of the mice isassessed by scoring the percent of mice living each day post treatment.

Since receptor tyrosine kinases frequently regulate cellular behaviorsthat are aberrant in tumors, EphA4 may provide a potential therapeutictarget for the antibody-based treatment of pancreatic cancer.Antibody-mediated aggregation of another Eph receptor family member,EphA2, decreases malignant potential of cancer cells (reviewed in Kinchand Carles-Kinch, Clinical and Experimental Metastasis, 20:59, 2003). Totest if EphA4-targeting would result in a similar outcome, several scFvfragments that bind human EphA4 were tested. EphA4 aggregation wasmediated by crosslinking an EphA4-specific scFv, using a secondaryantibody. EphA4 antibody treatment of a pancreatic carcinoma cell line,ASPC1, resulted in multiple biological and biochemical consequences.First, cell adhesions to the extracellular matrix decreased (FIG. 1),which is significant since these types of adhesions are sites ofintracellular signaling that promote cell growth, survival, migrationand invasion (Burridge and Chrzanowska-Wodnicka, Annu. Rev. Cell Dev.Biol., 12: 463, 1996; Parsons, Curr. Opin. Cell Biol., 8:146, 1996).Secondly, EphA4 antibody-mediated aggregation inhibitedanchorage-independent growth of ASPC1 cells, as measured using soft agarcolonization assays (FIG. 3). Third, EphA4 activation resulted in thetyrosine phosphorylation of EphA4 and an EphA4-associated 75 kDaprotein. Taken together, these data suggest that antibody-mediatedactivation of EphA4 initiates a signaling pathway that results in thereduced malignant potential of pancreatic carcinoma cells.

In another line of experiments, EphA4 upregulation promotesanchorage-independent growth of MCF-7 mammary carcinoma cell line to beless dependent on estrogen for growth. MCF-7 cells, which normallyexpress very low levels of EphA4, were transfected, and stableexpressors of EphA4 were isolated (EphA4-2 and EphA4-3). During softagar assays with estrogen, MCF-7-EphA4 cells demonstrated a 2.4 foldincrease in anchorage independent growth compared to avector-transfected control (FIG. 4). However in the absence of estrogen,EphA4-expressing cells had a 8.5 fold increase in growth compared to thesame controls. These data suggest that EphA4-overexpressing cells areless dependent on growth cues (such as matrix attachment and growthfactors) than cells without EphA4. The results of these experiments alsosuggest that antibody-mediated aggregation of MCF-7-EphA4 cells canreverse the increase in malignant potential.

6.4. EphA4 RNA Levels are Increased in Pancreatic Tumor Tissue

EphA4 RNA levels were determined from patient samples to determine EphA4expression in cancerous tissue. As demonstrated in FIG. 5, EphA4 RNAlevels are increased in pancreatic tumor tissue relative to normaltissue. The bar labeled “Normal” (A) shows EphA4 RNA levels inpathologically normal pancreatic tissue from a 52-year old male patient.“Stage 4 Primary” (B) shows EphA4 RNA levels in pancreatic tissuedisplaying Stage 4A ductal pancreatic adenocarcinoma from the same52-year old male patient from which the tissue in bar A was sampled.“Stage 2 Primary” (C) shows EphA4 RNA levels in pancreatic tissuedisplaying Stage 2A ductal pancreatic adenocarcinoma from a 72-year oldmale patient. “Met” (D) shows EphA4 RNA levels in lymph node tissue of a71-year old female patient displaying Stage 2B metastatic pancreaticadenocarcinoma. “Met” (E) shows EphA4 RNA levels in omentum tissue of a57-year old female patient displaying Stage 4 metastatic pancreaticadenocarcinoma. “Met” (F) shows EphA4 RNA levels in liver tissue of a45-year old female patient displaying metastatic pancreaticadenocarcinoma. From these samples, it is evident that EphA4 expressionis increased in cancerous and metastatic tissue relative to controltissue.

6.5. Treatment of Patients with Metastatic Cancer

A study is designed to assess pharmacokinetics and safety of monoclonalantibodies of the invention in patients with metastatic breast cancer.Patients currently receiving treatment are permitted to continue thesemedications.

Patients are administered a single IV dose of a monoclonal antibody ofthe invention and then, beginning 4 weeks later, are analyzed followingadministration of repeated weekly IV doses at the same dose over aperiod of 12 weeks. The safety of treatment with the monoclonal antibodyof the invention is assessed as well as potential changes in diseaseactivity over 26 weeks of IV dosing. Different groups of patients aretreated and evaluated similarly but receive doses of 1 mg/kg, 2 mg/kg, 4mg/kg, or 8 mg/kg.

Monoclonal antibodies of the invention are formulated at 5 mg/ml and 10mg/ml for IV injection. A formulation of 80 mg/ml is required forrepeated subcutaneous administration. The monoclonal antibodies of theinvention are also formulated at 100 mg/ml for administration for thepurposes of the study.

Changes are measured or determined by the progression of tumor growth.

6.6. Kinetic Analysis of EphA4 Antibodies

The surface plasmon resonance-based BIACORE™ assay is used to measurethe K_(off) rates of the monoclonal antibodies of the invention. IgGpresent in the hybridoma supernatant is used for measurement.

Immobilization of EphA4

EphA4 fusion protein is immobilized to a surface on a CM5 sensorchipusing a standard amine (70 μl of a 1:1 mix of NHS/EDC) couplingchemistry. Briefly, a 400 nM solution of EphA4 fusion protein in 10 mMNaOAc, pH4, is then injected over the activated surface to a density of1000-1100 RU's. Unused reactive esters are subsequently “capped” with a70 μl injection of 1M Et-NH2. Similarly, an activated and “capped”control surface is prepared on the same sensor chip without protein toserve as a reference surface.

Binding Experiments

A 250 μl injection of each of the EphA4 hybridoma supernatants is madeover both the EphA4 fusion protein and control surfaces, and the bindingresponses are recorded. These supernatants are used undiluted. Followingeach injection, at least 10 min. of dissociation phase data iscollected. Purified EphA4 monoclonal antibody is prepared to serve as apositive control (at 1 μg, 5 μg and 25 μg per 250 μl of growth medium).A negative control monoclonal antibody that does not bind EphA4 is alsoprepared at 5 μg/250 μl growth medium. Control injections of growthmedium across these surfaces are also made. Following each bindingcycle, the EphA4 fusion protein surface is regenerated with a single 1min. pulse (injection) of 1M NaCl-50 mM NaOH.

Data Evaluation

The binding data are corrected by subtracting out both artifactual noise(blank medium injections) and non-specific binding (control surface), ina technique known as “double-referencing.” Thus the sensorgram overlaysrepresent “net” binding curves. EphA4 antibodies have slow K_(off)rates.

7. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1-78. (canceled)
 79. A method of monitoring the efficacy of therapy forcancer in a patient known to or suspected to have cancer, said methodcomprising: a. contacting cells of said patient with an EphA4 antibodythat is an EphA4 agonistic antibody, an EphA4 cancer cell phenotypeinhibiting antibody, an exposed EphA4 epitope antibody under conditionsappropriate for antibody-EphA4 binding; and b. measuring EphA4 antibodybinding to said cells, wherein detecting a higher EphA4 antibody bindinglevel than in a control indicates that the patient has cancer.
 80. Themethod of claim 79 wherein said cells are from whole blood, sputum,urine, serum or fine needle aspirates of tumor cell tissue.
 81. Themethod of claim 79 wherein said cells are in frozen or fixed tissue orcells from said patient.
 82. The method of claim 79 wherein saiddetecting comprises imaging of said EphA4 antibody binding in saidpatient.
 83. The method of claim 79 wherein said patient has metastaticcancer.
 84. The method of claim 79 wherein said EphA4 antibody is anexposed EphA4 epitope antibody.
 85. An EphA4 antibody or antibodyfragment that is an EphA4 agonistic antibody, an EphA4 cancer cellphenotype inhibiting antibody, or an exposed EphA4 epitope antibodywherein the variable heavy chain and/or variable light chain amino acidsequence possesses at least 90% amino acid sequence identity with thevariable heavy chain or light chain amino acid sequence of EA44, ascontained in SEQ IDs NO: 4 and 8, respectively.
 86. The EphA4 antibodyof claim 85 wherein at least three CDRs of said variable light chain andvariable heavy chain are identical to the corresponding CDRs in EA44.87. The EphA4 antibody of claim 85 wherein at least four of its CDRs areidentical to the corresponding CDRs in EA44.
 88. The EphA4 antibody ofclaim 85 wherein at least five of its CDRS are identical to thecorresponding CDRs in EA44.
 89. The EphA4 antibody of claim 85 whereinall six of its CDRs are identical to the corresponding CDRs of EA44. 90.A cell line which produces an antibody of claim 85.